Detection of target nucleic acid sequences by PTO Cleavage and Extension assay

ABSTRACT

The present invention relates to the detection of a target nucleic acid sequence by a PTOCE (PTO Cleavage and Extension) assay. The present invention detects a target nucleic acid sequence in which the PTO (Probing and Tagging Oligonucleotide) hybridized with the target nucleic acid sequence is cleaved to release a fragment and the fragment is hybridized with the CTO (Capturing and Templating Oligonucleotide) to form an extended duplex, followed by detecting the presence of the extended duplex. The extended duplex provides signals (generation, increase, extinguishment or decrease of signals) from labels indicating the presence of the extended duplex and has adjustable T m  value, which are well adoptable for detection of the presence of the target nucleic acid sequence.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of and claims priority to U.S.application Ser. No. 13/702,546, filed Dec. 6, 2012, which has nowissued as U.S. Pat. No. 8,809,239 and which claims the priority ofPCT/KR2012/000287, filed on Jan. 11, 2012, which claims the benefit ofpriority to Korean Application Nos. 10-2011-0002840, filed Jan. 11,2011; 10-2011-0023465, filed Mar. 16, 2011; and PCT Application No,PCT/KR2011/004452, filed Jun. 17, 2011, the entire contents of each ofwhich are hereby incorporated in total by reference.

SEQUENCE

This application incorporates by reference the Sequence Listingcontained in an ASCII text file named “14-337493_ST25.txt” submitted viaEFS-Web. The text file was created on Sep. 23, 2014, and is 5.25 kb insize.

BACKGROUND OF THE INVENTION

Field of the Invention

The present invention relates to the detection of a target nucleic acidsequence by a PTOCE (PTO Cleavage and Extension) assay.

Description of the Related Art

DNA hybridization is a fundamental process in molecular biology and isaffected by ionic strength, base composition, length of fragment towhich the nucleic acid has been reduced, the degree of mismatching, andthe presence of denaturing agents. DNA hybridization-based technologieswould be a very useful tool in specific nucleic acid sequencedetermination and clearly be valuable in clinical diagnosis, geneticresearch, and forensic laboratory analysis.

However, the conventional methods and processes depending mostly onhybridization are very likely to produce false positive results due tonon-specific hybridization between probes and non-target sequences.Therefore, there remain problems to be solved for improving theirreliability.

Besides probe hybridization processes, several approaches usingadditional enzymatic reactions, for example, TaqMan™ probe method, havebeen suggested.

In TaqMan™ probe method, the labeled probe hybridized with a targetnucleic acid sequence is cleaved by a 5′ nuclease activity of anupstream primer-dependent DNA polymerase, generating a signal indicatingthe presence of a target sequence (U.S. Pat. Nos. 5,210,015, 5,538,848and 6,326,145). The TaqMan™ probe method suggests two approaches forsignal generation: polymerization-dependent cleavage andpolymerization-independent cleavage. In polymerization-dependentcleavage, extension of the upstream primer must occur before a nucleicacid polymerase encounters the 5′-end of the labeled probe. As theextension reaction continues, the polymerase progressively cleaves the5′-end of the labeled probe. In polymerization-independent cleavage, theupstream primer and the labeled probe are hybridized with a targetnucleic acid sequence in close proximity such that binding of thenucleic acid polymerase to the 3′-end of the upstream primer puts it incontact with the 5′-end of the labeled probe to release the label. Inaddition, the TaqMan™ probe method discloses that the labeled probe atits 5′-end having a 5′-tail region not-hybridizable with a targetsequence is also cleaved to form a fragment comprising the 5′-tailregion.

There have been reported some methods in which a probe having a 5′-tailregion non-complementary to a target sequence is cleaved by 5′ nucleaseto release a fragment comprising the 5′-tail region.

For instance, U.S. Pat. No. 5,691,142 discloses a cleavage structure tobe digested by 5′ nuclease activity of DNA polymerase. The cleavagestructure is exemplified in which an oligonucleotide comprising a 5′portion non-complementary to and a 3′ portion complementary to atemplate is hybridized with the template and an upstream oligonucleotideis hybridized with the template in close proximity. The cleavagestructure is cleaved by DNA polymerase having 5′ nuclease activity ormodified DNA polymerase with reduced synthetic activity to release the5′ portion non-complementary to the template. The released 5′ portion isthen hybridized with an oligonucleotide having a hairpin structure toform a cleavage structure, thereby inducing progressive, cleavagereactions to detect a target sequence.

U.S. Pat. No. 7,381,532 discloses a process in which the cleavagestructure having the upstream oligonucleotide with blocked 3′-end iscleaved by DNA polymerase having 5′ nuclease activity or FEN nuclease torelease non-complementary 5′ flap region and the released 5′ flap regionis detected by size analysis or interactive dual label. U.S. Pat. No.6,893,819 discloses that detectable released flaps are produced by anucleic acid synthesis dependent, flap-mediated sequential amplificationmethod. In this method, a released flap from a first cleavage structurecleaves, in a nucleic acid synthesis dependent manner, a second cleavagestructure to release a flap from the second cleavage structure and therelease flaps are detected.

By hybridization of fluorescence-labeled probes in a liquid phase, aplurality of target nucleic acid sequences may be simultaneouslydetected using even a single type of a fluorescent label by meltingcurve analysis. However, the conventional technologies for detection oftarget sequences by 5′ nuclease-mediated cleavage of interactive-duallabeled probes require different types of fluorescent labels fordifferent target sequences in multiplex target detection, which limitsthe number of target sequences to be detected due to limitation of thenumber of types of fluorescent labels.

U.S. Pat. Appln. Pub. 2008-0241838 discloses a target detection methodusing cleavage of a probe having a 5′ portion non-complementary to atarget nucleic acid sequence and hybridization of a capture probe. Alabel is positioned on the non complementary 5′ portion. The labeledprobe hybridized with the target sequence is cleaved to release afragment, after which the fragment is then hybridized with the captureprobe to detect the presence of the target sequence. In this method, itis necessary that an uncleaved/intact probe is not hybridized with thecapture probe. For that, the capture probe having a shorter length hasto be immobilized onto a solid substrate. However, such a limitationresults in lower efficiency of hybridization on a solid substrate andalso in difficulties in optimization of reaction conditions.

Therefore, there remain long-felt needs in the art to develop novelapproaches for detection of a target sequence, preferably multipletarget sequences, in a liquid phase and on a solid phase by not onlyhybridization but also enzymatic reactions such as 5 nucleolyticreaction in a more convenient, reliable and reproducible manner.Furthermore, a novel target detection method not limited by the numberof types of labels (particularly, fluorescent labels) is also needed inthe art.

Throughout this application, various patents and publications arereferenced and citations are provided in parentheses. The disclosure ofthese patents and publications in their entities are hereby incorporatedby references into this application in order to more fully describe thisinvention and the state of the art to which this invention pertains.

SUMMARY OF THE INVENTION

The present inventors have made intensive researches to develop novelapproaches to detect target sequences with more improved accuracy andconvenience, inter alia, in a multiplex manner. As a result, we haveestablished novel protocols for detection of target sequences, in whichtarget detection is accomplished by probe hybridization, enzymatic probecleavage, extension and detection of an extended duplex. The presentprotocols are well adopted to liquid phase reactions as well as solidphase reactions, and ensure detection of multiple target sequences withmore improved accuracy and convenience.

Therefore, it is an object of this invention to provide a method fordetecting a target nucleic acid sequence from a DNA or a mixture ofnucleic acids by a PTOCE (PTO Cleavage and Extension) assay.

It is another object of this invention to provide a kit for detecting atarget nucleic acid sequence from a DNA or a mixture of nucleic acids bya PTOCE (PTO Cleavage and Extension) assay.

Other objects and advantages of the present invention will becomeapparent from the detailed description to follow taken in conjugationwith the appended claims and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the schematic structures of PTO (Probing and TaggingOligonucleotide) and CTO (Capturing and Templating Oligonucleotide) usedin PTO cleavage and extension assay (PTOCE assay). Preferably, the3′-ends of the PTO and CTO are blocked to prohibit their extension.

FIG. 2 represents schematically PTOCE assay comprising melting analysis.CTO has a reporter molecule and a quencher molecule at its templatingportion.

FIG. 3 represents schematically PTOCE assay comprising melting analysis.CTO has a reporter molecule at its templating portion. The reportermolecule is required to show different signal intensity depending on itspresence on a single-stranded form or a double-stranded form.

FIG. 4 represents schematically PTOCE assay comprising melting analysis.CTO has an iso-dC residue and a reporter molecule at its templatingportion. Quencher-iso-dGTP is incorporated into the extended duplexduring extension reaction.

FIG. 5 represents schematically PTOCE assay comprising melting analysis.PTO has a reporter molecule at its 5′-tagging portion and CTO has aniso-dC residue at its templating portion. Quencher-iso-dGTP isincorporated into the extended duplex during extension reaction.

FIG. 6 represents schematically PTOCE assay comprising melting analysis.PTO has a reporter molecule and a quencher molecule at its 5′-taggingportion.

FIG. 7 represents schematically PTOCE assay comprising melting analysis.PTO has a reporter molecule at its 5′-tagging portion. The reportermolecule is required to show different signal intensity depending on itspresence on a single-stranded form or a double-stranded form.

FIG. 8 represents schematically PTOCE assay comprising melting analysis.PTO has a quencher molecule at its 5′-tagging portion and CTO has areporter molecule at its capturing portion.

FIG. 9 represents schematically PTOCE assay comprising detection at apre determined temperature. CTO has a reporter molecule and a quenchermolecule at its templating portion. CTO is immobilized on a solidsubstrate through its 3′-end.

FIG. 10 represents schematically PTOCE assay comprising detection at apre-determined temperature. A reporter-labeled dATP is incorporated intothe extended duplex during extension reaction. CTO is immobilized on asolid substrate through its 3′-end.

FIG. 11 represents schematically PTOCE assay comprising detection at apre-determined temperature. CTO has an iso-dC residue at its templatingportion and a reporter-iso-dGTP is incorporated into the extended duplexduring extension reaction. CTO is immobilized on a solid substratethrough its 3′-end.

FIG. 12 represents schematically PTOCE assay comprising detection at apre-determined temperature. PTO has a reporter molecule at its5′-tagging portion. CTO is immobilized on a solid substrate through its5′-end.

FIG. 13 represents schematically PTOCE assay comprising detection at apre-determined temperature with an intercalating dye. CTO is immobilizedon a solid substrate through its 5′-end.

FIG. 14 shows the results of the detection of Neisseria gonorrhoeae geneby PTOCE assay comprising melting analysis. CTO has a reporter moleculeand a quencher molecule at its templating portion.

FIG. 15 shows the results of the detection of Neisseria gonorrhoeae geneby PTOCE assay comprising melting analysis. PTO has a quencher moleculeat its 5′-end and CTO has a reporter molecule at its 3′-end.

FIG. 16 shows the results that Tm values of extended duplexes areadjusted by CTO sequences.

FIG. 17A and FIG. 17B show the results of the detection of Neisseriagonorrhoeae gene by PTOCE assay with PCR amplification. CTO has areporter molecule and a quencher molecule at its templating portion.FIG. 17A shows the results of PTOCE assay comprising real-time PCRdetection and FIG. 17B shows the results of PTOCE assay comprisingpost-PCR melting analysis.

FIG. 18A and FIG. 18B show the results of the detection of Neisseriagonorrhoeae gene by PTOCE assay with PCR amplification. CTO has aniso-dC residue and a reporter molecule at its 5′-end. Quencher-iso-dGTPis incorporated into the extended duplex during extension reaction. FIG.18A shows the results of PTOCE assay comprising real-time PCR detectionand FIG. 18B shows the results of PTOCE assay comprising post-PCRmelting analysis.

FIG. 19A and FIG. 19B show the results of the detection of Neisseriagonorrhoeae gene by PTOCE assay with PCR amplification. PTO has aquencher molecule at its 5′-end and CTO has a reporter molecule at its3′-end. FIG. 19A shows the results of PTOCE assay comprising real-timePCR detection and FIG. 19B show the results of PTOCE assay comprisingpost-PCR melting analysis.

FIG. 20 shows the results of the simultaneous detection of Neisseriagonorrhoeae (NG) gene and Staphylococcus aureus (SA) gene by PTOCE assaycomprising post PCR melting analysis. CTO has a reporter molecule and aquencher molecule at its templating portion.

FIG. 21A and FIG. 21B show the results of the detection of Neisseriagonorrhoeae gene by PTOCE assay comprising melting analysis onmicroarray. CTO is immobilized through its 5′-end. PTO has a reportermolecule at its 5′-tagging portion. FIG. 21A depicts the fluorescentimage depending on temperature on microarray. FIG. 21B depicts thefluorescent intensity depending on temperature on microarray.

FIG. 22A and FIG. 22B show the results of the detection of Neisseriagonorrhoeae gene by PTOCE assay comprising real-time detection at apre-determined temperature on microarray. CTO is immobilized through its5′-end. PTO has a reporter molecule at its 5′-tagging portion. FIG. 22Adepicts the fluorescent images depending on cycle numbers on microarray.FIG. 22B depicts the change of fluorescence intensity depending on cyclenumbers on microarray.

FIG. 23A and FIG. 23B show the results of the detection of Neisseriagonorrhoeae gene by PTOCE assay comprising real-time detection at apre-determined temperature on microarray. CTO is immobilized through its3′-end and has a reporter molecule and a quencher molecule at itstemplating portion. FIG. 23A depicts the fluorescent images depending oncycle numbers on microarray. FIG. 23B depicts the change of fluorescenceintensity depending on cycle numbers on microarray.

FIG. 24 shows the results of the single or multiple target detection byPTOCE assay comprising end point detection at a pre-determinedtemperature on microarray. CTO is immobilized through its 5′-end. PTOhas a reporter molecule at its 5′-tagging portion. Neisseria gonorrhoeae(NG) gene and Staphylococcus aureus (SA) gene were used as targetnucleic acid sequences.

DETAILED DESCRIPTION OF THIS INVENTION

The present invention is directed to a novel method for detecting atarget nucleic acid sequence by a PTOCE (PTO Cleavage and Extension)assay and a kit for detecting a target nucleic acid sequence by a PTOCEassay.

The present invention involves not only hybridization reactions but alsoenzymatic reactions that occur depending on the presence of a targetnucleic acid sequence.

I. Target Detection Process by PTOCE Comprising Melting Analysis

In one aspect of the present invention, there is provided a method fordetecting a target nucleic acid sequence from a DNA or a mixture ofnucleic acids by a PTOCE (PTO Cleavage and Extension) assay, comprising:

-   -   (a) hybridizing the target nucleic acid sequence with an        upstream oligonucleotide and a PTO (Probing and Tagging        Oligonucleotide), wherein the upstream oligonucleotide comprises        a hybridizing nucleotide sequence complementary to the target        nucleic acid sequence; the PTO comprises (i) a 3′-targeting        portion comprising a hybridizing nucleotide sequence        complementary to the target nucleic acid sequence and (ii) a        5′-tagging portion comprising a nucleotide sequence        non-complementary to the target nucleic acid sequence; wherein        the 3′-targeting portion is hybridized with the target nucleic        acid sequence and the 5′-tagging portion is not hybridized with        the target nucleic acid sequence; the upstream oligonucleotide        is located upstream of the PTO;    -   (b) contacting the resultant of the step (a) to an enzyme having        a 5′ nuclease activity under conditions for cleavage of the PTO;        wherein the upstream oligonucleotide or its extended strand        induces cleavage of the PTO by the enzyme having the 5′ nuclease        activity such that the cleavage releases a fragment comprising        the 5′-tagging portion or a part of the 5′-tagging portion of        the PTO;    -   (c) hybridizing the fragment released from the PTO with a CTO        (Capturing and Templating Oligonucleotide); wherein the CTO        comprises in a 3′ to 5′ direction (i) a capturing portion        comprising a nucleotide sequence complementary to the 5′-tagging        portion or a part of the 5′-tagging portion of the PTO and (ii)        a templating portion comprising a nucleotide sequence        non-complementary to the 5′-tagging portion and the 3′-targeting        portion of the PTO; wherein the fragment released from the PTO        is hybridized with the capturing portion of the CTO;    -   (d) performing an extension reaction using the resultant of the        step (c) and a template-dependent nucleic acid polymerase;        wherein the fragment hybridized with the capturing portion of        the CTO is extended and an extended duplex is formed; wherein        the extended duplex has a T_(m) value adjustable by (i) a        sequence and/or length of the fragment, (ii) a sequence and/or        length of the CTO or (iii) the sequence and/or length of the        fragment and the sequence and/or length of the CTO;    -   (e) melting the extended duplex over a range of temperatures to        give a target signal indicative of the presence of the extended        duplex; wherein the target signal is provided by (i) at least        one label linked to the fragment and/or the CTO, (ii) a label        incorporated into the extended duplex during the extension        reaction, (iii) a label incorporated into the extended duplex        during the extension reaction and a label linked to the fragment        and/or the CTO, or (iv) an intercalating label; and    -   (f) detecting the extended duplex by measuring the target        signal; whereby the presence of the extended duplex indicates        the presence of the target nucleic acid sequence.

The present inventors have made intensive researches to develop novelapproaches to detect target sequences with more improved accuracy andconvenience, inter alia, in a multiplex manner. As a result, we haveestablished novel protocols for detection of target sequences, in whichtarget detection is accomplished by probe hybridization, enzymatic probecleavage, extension and detection of an extended duplex. The presentprotocols are well adopted to liquid phase reactions as well as solidphase reactions, and ensure detection of multiple target sequences withmore improved accuracy and convenience.

The present invention employs successive events followed by probehybridization; cleavage of PTO (Probing and Tagging Oligonucleotide) andextension; formation of a target-dependent extended duplex; anddetection of the extended duplex. Therefore, it is named as a PTOCE (PTOCleavage and Extension) assay.

In the present invention, the extended duplex is characterized to have alabel(s) providing a signal indicating the presence of the extendedduplex by melting analysis or by detection at a pre-determinedtemperature. Furthermore, the extended duplex is characterized to havean adjustable T_(m) value, which plays a critical role in multipletarget detection or discrimination from non-target signal.

As the extended duplex is produced only if the target nucleic acidexists, the presence of the extended duplex indicates the presence ofthe target nucleic acid.

The PTOCE assay comprising melting analysis will be described in moredetail as follows:

Step (a): Hybridization of an Upstream Oligonucleotide and a PTO with aTarget Nucleic Acid Sequence

According to the present invention, a target nucleic acid sequence isfirst hybridized with an upstream oligonucleotide and a PTO (Probing andTagging Oligonucleotide).

The term used herein “target nucleic acid”, “target nucleic acidsequence” or “target sequence” refers to a nucleic acid sequence ofinterest for detection, which is annealed to or hybridized with a probeor primer under hybridization, annealing or amplifying conditions.

The term used herein “probe” refers to a single-stranded nucleic acidmolecule comprising a portion or portions that are substantiallycomplementary to a target nucleic acid sequence.

The term “primer” as used herein refers to an oligonucleotide, which iscapable of acting as a point of initiation of synthesis when placedunder conditions in which synthesis of primer extension product which iscomplementary to a nucleic acid strand (template) is induced, i.e., inthe presence of nucleotides and an agent for polymerization, such as DNApolymerase, and at a suitable temperature and pH.

Preferably, the probe and primer are single-stranded deoxyribonucleotidemolecules. The probes or primers used in this invention may be comprisedof naturally occurring dNMP (i.e., dAMP, dGM, dCMP and dTMP), modifiednucleotide, or non natural nucleotide. The probes or primers may alsoinclude ribonucleotides.

The primer must be sufficiently long to prime the synthesis of extensionproducts in the presence of the agent for polymerization. The exactlength of the primers will depend on many factors, includingtemperature, application, and source of primer. The term “annealing” or“priming” as used herein refers to the apposition of anoligodeoxynucleotide or nucleic acid to a template nucleic acid, wherebythe apposition enables the polymerase to polymerize nucleotides into anucleic acid molecule which is complementary to the template nucleicacid or a portion thereof.

The term used “hybridizing” used herein refers to the formation of adouble-stranded nucleic acid from complementary single stranded nucleicacids. The hybridization may occur between two nucleic acid strandsperfectly matched or substantially matched with some mismatches. Thecomplementarity for hybridization may depend on hybridizationconditions, particularly temperature.

The hybridization of a target nucleic acid sequence with the upstreamoligonucleotide and the PTO may be carried out under suitablehybridization conditions routinely determined by optimizationprocedures. Conditions such as temperature, concentration of components,hybridization and washing times, buffer components, and their pH andionic strength may be varied depending on various factors, including thelength and GC content of oligonucleotide (upstream oligonucleotide andPTO) and the target nucleotide sequence. For instance, when a relativelyshort oligonucleotide is used, it is preferable that low stringentconditions are adopted. The detailed conditions for hybridization can befound in Joseph Sambrook, et al., Molecular Cloning, A LaboratoryManual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.(2001); and M. L. M. Anderson, Nucleic Acid Hybridization,Springer-Verlag New York Inc. N.Y. (1999).

There is no intended distinction between the terms “annealing” and“hybridizing”, and these terms will be used interchangeably.

The upstream oligonucleotide and PTO have hybridizing nucleotidesequences complementary to the target nucleic acid sequence. The term“complementary” is used herein to mean that primers or probes aresufficiently complementary to hybridize selectively to a target nucleicacid sequence under the designated annealing conditions or stringentconditions, encompassing the terms “substantially complementary” and“perfectly complementary”, preferably perfectly complementary.

The 5′-tagging portion of the PTO has a nucleotide sequence noncomplementary to the target nucleic acid sequence. The templatingportion of the CTO (Capturing and Templating Oligonucleotide) has anucleotide sequence non-complementary to the 5′-tagging portion and the3′-targeting portion of the PTO. The term “non-complementary” is usedherein to mean that primers or probes are sufficiently non-complementarynot to hybridize selectively to a target nucleic acid sequence under thedesignated annealing conditions or stringent conditions, encompassingthe terms “substantially non-complementary” and “perfectly noncomplementary”, preferably perfectly non-complementary.

The term used herein “PTO (Probing and Tagging Oligonucleotide)” meansan oligonucleotide comprising (I) a 3′-targeting portion serving as aprobe and (ii) a 5′-tagging portion with a nucleotide sequencenon-complementary to the target nucleic acid sequence, which isnucleolytically released from the PTO after hybridization with thetarget nucleic acid sequence. The 5′-tagging portion and the3′-targeting portion in the PTO have to be positioned in a 5′ to 3′order. The PTO is schematically illustrated in FIG. 1.

Preferably, the hybridization in step (a) is preformed under stringentconditions that the 3′-targeting portion is hybridized with the targetnucleic acid sequence and the 5′-tagging portion is not hybridized withthe target nucleic acid sequence.

The PTO does not require any specific lengths. For example, the lengthof the PTO may be 15-150 nucleotides, 15-100 nucleotides, 15-80nucleotides, 15-60 nucleotides, 15-40 nucleotides, 20-150 nucleotides,20-100 nucleotides, 20-80 nucleotides, 20-60 nucleotides, 20-50nucleotides, 30450 nucleotides, 30-100 nucleotides, 30-80 nucleotides,30-60 nucleotides, 30-50 nucleotides, 35-100 nucleotides, 35-80nucleotides, 35-60 nucleotides, or 35-50 nucleotides. The 3′-targetingportion of the PTO may be in any lengths so long as it is specificallyhybridized with target nucleic acid sequences. For example, the3′-targeting portion of the PTO may be 10-100 nucleotides, 10-80nucleotides, 10-50 nucleotides, 10-40 nucleotides, 10-30 nucleotides,15-100 nucleotides, 15-80 nucleotides, 15-50 nucleotides, 15-40nucleotides, 15-30 nucleotides, 20-100 nucleotides, 20-80 nucleotides,20-50 nucleotides, 20-40 nucleotides or 20-30 nucleotides in length. The5′-tagging portion may be in any lengths so long as it is specificallyhybridized with the templating portion of the CTO and then extended. Forinstance, the 5′-tagging portion of the PTO may be 5-50 nucleotides,5-40 nucleotides, 5-30 nucleotides, 5-20 nucleotides, 10-50 nucleotides,10-40 nucleotides, 10-30 nucleotides, 10-20 nucleotides, 15-50nucleotides, 15-40 nucleotides, 15-30 nucleotides or 15-20 nucleotidesin length.

The 3′-end of the PTO may have a 3′-OH terminal. Preferably, the 3′-endof the PTO is “blocked” to prohibit its extension.

The blocking may be achieved in accordance with conventional methods.For instance, the blocking may be performed by adding to the 3′-hydroxylgroup of the last nucleotide a chemical moiety such as biotin, labels, aphosphate group, alkyl group, non-nucleotide linker, phosphorothioate oralkane-diol. Alternatively, the blocking may be carried out by removingthe 3′-hydroxyl group of the last nucleotide or using a nucleotide withno 3′-hydroxyl group such as dideoxynucleotide.

Alternatively, the PTO may be designed to have a hairpin structure.

The non-hybridization between the 5′-tagging portion of the PTO and thetarget nucleic acid sequence refers to non-formation of a stabledouble-strand between them under certain hybridization conditions.According to a preferred embodiment, the 5′-tagging portion of the PTOnot involved in the hybridization with the target nucleic acid sequenceforms a single-strand.

The upstream oligonucleotide is located upstream of the PTO.

In addition, the upstream oligonucleotide or its extended strandhybridized with the target nucleic acid sequence induces cleavage of thePTO by an enzyme having a 5′ nuclease activity.

The induction of the PTO cleavage by the upstream oligonucleotide may beaccomplished by two fashions: (i) upstream oligonucleotideextension-independent cleavage induction; and (ii) upstreamoligonucleotide extension-dependent cleavage induction.

Where the upstream oligonucleotide is positioned adjacently to the PTOsufficient to induce the PTO cleavage by an enzyme having a 5′ nucleaseactivity, the enzyme bound to the upstream oligonucleotide digests thePTO with no extension reaction. In contrast, where the upstreamoligonucleotide is positioned distantly to the PTO, an enzyme having apolymerase activity (e.g., template-dependent polymerase) catalyzesextension of the upstream oligonucleotide (e.g., upstream primer) and anenzyme having a 5′ nuclease activity bound to the extended productdigests the PTO.

Therefore, the upstream oligonucleotide may be located relatively to thePTO in two fashions. The upstream oligonucleotide may be locatedadjacently to the PTO sufficient to induce the PTO cleavage in anextension-independent manner. Alternatively, the upstreamoligonucleotide may be located distantly to the PTO sufficient to inducethe PTO cleavage in an extension-dependent manner.

The term used herein “adjacent” with referring to positions or locationsmeans that the upstream oligonucleotide is located adjacently to the3′-targeting portion of the PTO to form a nick. Also, the term meansthat the upstream oligonucleotide is located 1-30 nucleotides, 1-20nucleotides or 1-15 nucleotides apart from the 3′-targeting portion ofthe PTO.

The term used herein “distant” with referring to positions or locationsincludes any positions or locations sufficient to ensure extensionreactions.

According to a preferred embodiment, the upstream oligonucleotide islocated distantly to the PTO sufficient to induce the PTO cleavage in anextension-dependent manner.

According to a preferred embodiment, the upstream oligonucleotide is anupstream primer or an upstream probe. The upstream primer is suitable inan extension-independent cleavage induction or an extension-dependentcleavage, and the upstream probe is suitable in an extension-independentcleavage induction.

Alternatively, the upstream oligonucleotide may have apartial-overlapped sequence with the 5′-part of the 3′-targeting portionof the PTO. Preferably, the overlapped sequence is 1-10 nucleotides,more preferably 1-5 nucleotides, still more preferably 1-3 nucleotidesin length. Where the upstream oligonucleotide has a partial overlappedsequence with the 5′-part of the 3′-targeting portion of the PTO, the3′-targeting portion is partially digested along with the 5′-taggingportion in the cleavage reaction of the step (b). In addition, theoverlapped sequence permits to cleave a desired site of the 3′-targetingportion.

According to a preferred embodiment, the upstream primer induces throughits extended strand the cleavage of the PTO by the enzyme having the 5′nuclease activity.

The conventional technologies for cleavage reactions by upstreamoligonucleotides may be applied to the present invention, so long as theupstream oligonucleotide induces cleavage of the PTO hybridized with thetarget nucleic acid sequence to release a fragment comprising the5′-tagging portion or a part of the 5′-tagging portion of the PTO. Forexample, U.S. Pat. Nos. 5,210,015, 5,487,972, 5,691,142, 5,994,069 and7,381,532 and U.S. Appln. Pub. No. 2008-0241838 may be applied to thepresent invention.

According to a preferred embodiment, the method is performed in thepresence of a downstream primer. The downstream primer generatesadditionally a target nucleic acid sequence to be hybridized with thePTO, enhancing sensitivity in a target detection.

According to a preferred embodiment, when the upstream primer and thedownstream primer are used, a template-dependent nucleic acid polymeraseis additionally employed for extension of the primers.

According to a preferred embodiment, the upstream oligonucleotide(upstream primer or upstream probe), the downstream primer and/or5′-tagging portion of the PTO have a dual priming oligonucleotide (DPO)structure developed by the present inventor. The oligonucleotides havingthe DPO structure show significantly improved target specificitycompared with conventional primers and probes (see WO 2006/095981; Chunet al., Dual priming oligonucleotide system for the multiplex detectionof respiratory viruses and SNP genotyping of CYP2C19 gene, Nucleic AcidResearch, 35:6e40(2007)).

According to a preferred embodiment, the 3′-targeting portion of the PTOhas a modified dual specificity oligonucleotide (mDSO) structuredeveloped by the present inventor. The modified dual specificityoligonucleotide (mDSO) structure shows significantly improved targetspecificity compared with conventional probes (see WO 2011/028041)

Step (b): Release of a Fragment from the PTO

Afterwards, the resultant of the step (a) is contacted to an enzymehaving a 5′ nuclease activity under conditions for cleavage of the PTO.The PTO hybridized with the target nucleic acid sequence is digested bythe enzyme having the 5′ nuclease activity to release a fragmentcomprising the 5′-tagging portion or a part of the 5′-tagging portion ofthe PTO.

The term used herein “conditions for cleavage of the PTO” meansconditions sufficient to digest the PTO hybridized with the targetnucleic acid sequence by the enzyme having the 5′ nuclease activity,such as temperature, pH, ionic strength, buffer, length and sequence ofoligonucleotides and enzymes. For example, when Taq DNA polymerase isused as the enzyme having the 5′ nuclease activity, the conditions forcleavage of the PTO include Tris-HCl buffer, KCl, MgCl₂ and temperature.

When the PTO is hybridized with the target nucleic acid sequence, its3′-targeting portion is involved in the hybridization and the 5′-taggingportion forms a single-strand with no hybridization with the targetnucleic acid sequence (see FIG. 2). As such, an oligonucleotidecomprising both single-stranded and double-stranded structures may bedigested using an enzyme having a 5′ nuclease activity by a variety oftechnologies known to one of skill in the art.

The cleavage sites of the PTO are varied depending on the type ofupstream oligonucleotides (upstream probe or upstream primer),hybridization sites of upstream oligonucleotides and cleavage conditions(see U.S. Pat. Nos. 5,210,015, 5,487,972, 5,691,142, 5,994,069 and7,381,532 and U.S. Appln. Pub. No. 2008-0241838).

A multitude of conventional technologies may be employed for thecleavage reaction of the PTO, releasing a fragment comprising the5′-tagging portion or a part of the 5′-tagging portion.

Briefly, there may be three sites of cleavage in the step (b). Firstly,the cleavage site is a junction site between a hybridization portion ofthe PTO (3′-targeting portion) and a non-hybridization portion(5′-tagging portion). The second cleavage site is a site located severalnucleotides in a 3′-direction apart from the 3′-end of the 5′-taggingportion of the PTO. The second cleavage site is located at the 5′-endpart of the 3′-targeting portion of the PTO. The third cleavage site isa site located several nucleotides in a 5′-direction apart from the3′-end of the 5′-tagging portion of the PTO.

According to a preferred embodiment, the initial site for the cleavageof the PTO by the template-dependent polymerase having the 5′ nucleaseactivity upon extension of the upstream primer is a starting point ofthe double strand between the PTO and the target nucleic acid sequenceor a site 1-3 nucleotides apart from the starting point.

In this regard, the term used herein “a fragment comprising the5′-tagging portion or a part of the 5′-tagging portion of the PTO” inconjunction with cleavage of the PTO by the enzyme having the 5′nuclease activity is used to encompass (i) the 5′-tagging portion, (ii)the 5′-tagging portion and the 5′-end part of the 3′-targeting portionand (iii) a part of the 5′-tagging portion. In this application, theterm “a fragment comprising the 5′-tagging portion or a part of the5′-tagging portion of the PTO” may be also described as “PTO fragment”.

The term “part” used in conjunction with the PTO or CTO such as the partof the 5′-tagging portion of the PTO, the 5′-end part of the3′-targeting portion of the PTO and the 5′-end part of the capturingportion of the CTO refers to a nucleotide sequence composed of 1-40,1-30, 1-20, 1-15, 1-10 or 1-5 nucleotides, preferably 1, 2, 3 or 4nucleotides.

According to a preferred embodiment, the enzyme having the 5′ nucleaseactivity is DNA polymerase having a 5′ nuclease activity or FENnuclease, more preferably a thermostable DNA polymerase having a 5′nuclease activity or FEN nuclease.

A suitable DNA polymerase having a 5′ nuclease activity in thisinvention is a thermostable DNA polymerase obtained from a variety ofbacterial species, including Thermus aquaticus (Taq), Thermusthermophilus (Tth), Thermus filiformis, Thermus flavus, Thermococcusliteralis, Thermus antranikianii, Thermus caldophilus, Thermuschliarophilus, Thermus flavus, Thermus igniterrae, Thermus lacteus,Thermus oshimai, Thermus ruber, Thermus rubens, Thermus scotoductus,Thermus silvanus, Thermus species Z05, Thermus species sps 17, Thermusthermophilus, Thermotoga maritima, Thermotoga neapolitana, Thermosiphoafricanus, Thermococcus litoralis, Thermococcus barossi, Thermococcusgorgonarius, Thermotoga maritima, Thermotoga neapolitana,Thermosiphoafricanus, Pyrococcus woesei, Pyrococcus horikoshii,Pyrococcus abyssi, Pyrodictium occultum, Aquifex pyrophilus and Aquifexaeolieus. Most preferably, the thermostable DNA polymerase is Taqpolymerase.

Alternatively, the present invention may employ DNA polymerases having a5′ nuclease activity modified to have less polymerase activities.

The FEN (flap endonuclease) nuclease used is a 5′ flap-specificnuclease.

The FEN nuclease suitable in the present invention comprises FENnucleases obtained from a variety of bacterial species, includingSulfolobus solfataricus, Pyrobaculum aerophilum, Thermococcus litoralis,Archaeaglobus veneficus, Archaeaglobus profundus, Acidianus brierlyi,Acidianus ambivalens, Desulfurococcus amylolyticus, Desulfurococcusmobilis, Pyrodictium brockii, Thermococcus gorgonarius, Thermococcuszilligii, Methanopyrus kandleri, Methanococcus igneus, Pyrococcushorikoshii, Aeropyrum pernix, and Archaeaglobus veneficus.

Where the upstream primer is used in the step (a), it is preferable thatthe conditions for cleavage of the PTO comprise extension reaction ofthe upstream primer.

According to a preferred embodiment, the upstream primer is used in thestep (a), a template-dependent polymerase is used for extension of theupstream primer and the template-dependent polymerase is identical tothe enzyme having the 5′ nuclease activity.

Optionally, the upstream primer is used in the step (a), atemplate-dependent polymerase is used for extension of the upstreamprimer and the template-dependent polymerase is different from theenzyme having the 5′ nuclease activity.

Step (c): Hybridization of the Fragment Released from the PTO with CTO

The fragment released from the PTO is hybridized with a CTO (Capturingand Templating Oligonucleotide).

The CTO comprises in a 3′ to 5′ direction (i) a capturing portioncomprising a nucleotide sequence complementary to the 5′-tagging portionor a part of the 5′-tagging portion of the PTO and (ii) a templatingportion comprising a nucleotide sequence non-complementary to the5′-tagging portion and the 3′-targeting portion of the PTO.

The CTO is acted as a template for extension of the fragment releasedfrom the PTO. The fragment serving as a primer is hybridized with theCTO and extended to form an extended duplex.

The templating portion may comprise any sequence so long as it isnon-complementary to the 5′-tagging portion and the 3′-targeting portionof the PTO. Furthermore, the templating portion may comprise anysequence so long as it can be acted as a template for extension of thefragment released from the PTO.

As described above, when the fragment having the 5′-tagging portion ofthe PTO is released, it is preferred that the capturing portion of theCTO is designed to comprise a nucleotide sequence complementary to the5′-tagging portion. When the fragment having the 5′-tagging portion anda 5′-end part of the 3′-targeting portion is released, it is preferredthat the capturing portion of the CTO is designed to comprise anucleotide sequence complementary to the 5′-tagging portion and the5′-end part of the 3′-targeting portion. When the fragment having a partof the 5′-tagging portion of the PTO is released, it is preferred thatthe capturing portion of the CTO is designed to comprise a nucleotidesequence complementary to the part of the 5′-tagging portion.

Moreover, it is possible to design the capturing portion of the CTO withanticipating cleavage sites of the PTO. For example, where the capturingportion of the CTO is designed to comprise a nucleotide sequencecomplementary to the 5′-tagging portion, either the fragment having apart of the 5′-tagging portion or the fragment having the 5′-taggingportion can be hybridized with the capturing portion and then extended.Where the fragment comprising the 5′-tagging portion and a 5′-end partof the 3′-targeting portion is released, it may be hybridized with thecapturing portion of the CTO designed to comprise a nucleotide sequencecomplementary to the 5′-tagging portion and then successfully extendedalthough mismatch nucleotides are present at the 3′-end portion of thefragment. That is because primers can be extended depending on reactionconditions although its 3′-end contains some mismatch nucleotides (e.g.1-3 mismatch nucleotides).

When the fragment comprising the 5′-tagging portion and a 5′-end part ofthe 3′-targeting portion is released, the 5′-end part of the capturingportion of the CTO may be designed to have a nucleotide sequencecomplementary to the cleaved 5′-end part of the 3′-targeting portion,overcoming problems associated with mismatch nucleotides (see FIG. 1).

Preferably, the nucleotide sequence of the 5′-end part of the capturingportion of the CTO complementary to the cleaved 5′-end part of the3′-targeting portion may be selected depending on anticipated cleavagesites on the 3′-targeting portion of the PTO. It is preferable that thenucleotide sequence of the 5′-end part of the capturing portion of theCTO complementary to the cleaved 5′-end part of the 3′-targeting portionis 1-10 nucleotides, more preferably 1-5 nucleotides, still morepreferably 1-3 nucleotides.

The 3′-end of the CTO may comprise additional nucleotides not involvedin hybridization with the fragment. Moreover, the capturing portion ofthe CTO may comprise a nucleotide sequence complementary only to a partof the fragment (e.g., a part of the fragment containing its 3′-endportion) so long as it is stably hybridized with the fragment.

The term used “capturing portion comprising a nucleotide sequencecomplementary to the 5′-tagging portion or a part of the 5′-taggingportion” is described herein to encompass various designs andcompositions of the capturing portion of the CTO as discussed above.

The CTO may be designed to have a hairpin structure.

The length of the CTO may be widely varied. For example, the CTO is7-1000 nucleotides, 7-500 nucleotides, 7-300 nucleotides, 7-100nucleotides, 7-80 nucleotides, 7-60 nucleotides, 7-40 nucleotides,15-1000 nucleotides, 15-500 nucleotides, 15-300 nucleotides, 15-100nucleotides, 15-80 nucleotides, 15-60 nucleotides, 15-40 nucleotides,20-1000 nucleotides, 20-500 nucleotides, 20-300 nucleotides, 20-100nucleotides, 20-80 nucleotides, 20-60 nucleotides, 20-40 nucleotides,30-1000 nucleotides, 30-500 nucleotides, 30-300 nucleotides, 30-100nucleotides, 30-80 nucleotides, 30-60 nucleotides or 30-40 nucleotidesin length. The capturing portion of the CTO may have any length so longas it is specifically hybridized with the fragment released from thePTO. For example, the capturing portion of the CTO is 5-100 nucleotides,5-60 nucleotides, 5-40 nucleotides, 5-30 nucleotides, 5-20 nucleotides,10-100 nucleotides, 10-60 nucleotides, 10-40 nucleotides, 10-30nucleotides, 10-20 nucleotides, 15-100 nucleotides, 15-60 nucleotides,15-40 nucleotides, 15-30 nucleotides or 15-20 nucleotides in length. Thetemplating portion of the CTO may have any length so long as it can actas a template in extension of the fragment released from the PTO. Forexample, the templating portion of the CTO is 2-900 nucleotides, 2-400nucleotides, 2-300 nucleotides, 2-100 nucleotides, 2-80 nucleotides,2-60 nucleotides, 2-40 nucleotides, 2-20 nucleotides, 5-900 nucleotides,5-400 nucleotides, 5-300 nucleotides, 5-100 nucleotides, 5-80nucleotides, 5-60 nucleotides, 5-40 nucleotides, 5-30 nucleotides,10-900 nucleotides, 10-400 nucleotides, 10-300 nucleotides, 15-900nucleotides, 15-100 nucleotides, 15-80 nucleotides, 15-60 nucleotides,15-40 nucleotides or 15-20 nucleotides in length.

The 3′-end of the CTO may have a 3′-OH terminal. Preferably, the 3′-endof the CTO is blocked to prohibit its extension. The non-extendibleblocking of the CTO may be achieved in accordance with conventionalmethods. For instance, the blocking may be performed by adding to the3′-hydroxyl group of the last nucleotide of the CTO a chemical moietysuch as biotin, labels, a phosphate group, alkyl group, non-nucleotidelinker, phosphorothioate or alkane-diol. Alternatively, the blocking maybe carried out by removing the 3′-hydroxyl group of the last nucleotideor using a nucleotide with no 3′-hydroxyl group such asdideoxynucleotide.

The fragment released from the PTO is hybridized with the CTO, providinga form suitable in extension of the fragment. Although an undigested PTOis also hybridized with the capturing portion of the CTO through its5′-tagging portion, its 3′-targeting portion is not hybridized to theCTO which prohibits the formation of an extended duplex.

The hybridization in the step (c) can be described in detail withreferring to descriptions in the step (a).

Step (d): Extension of the Fragment

The extension reaction is carried out using the resultant of the step(c) and a template-dependent nucleic acid polymerase. The fragmenthybridized with the capturing portion of the CTO is extended to form anextended duplex. In contrast, uncleaved PTO hybridized with thecapturing portion of the CTO is not extended such that no extendedduplex is formed.

The term used herein “extended duplex” means a duplex formed byextension reaction in which the fragment hybridized with the capturingportion of the CTO is extended using the templating portion of the CTOas a template and the template-dependent nucleic acid polymerase.

The extended duplex has different T_(m) value from that of the hybridbetween the uncleaved PTO and the CTO.

Preferably, the extended duplex has higher T_(m) value than the hybridbetween the uncleaved PTO and the CTO.

The T_(m) value of the extended duplex is adjustable by (i) a sequenceand/or length of the fragment, (ii) a sequence and/or length of the CTOor (iii) the sequence and/or length of the fragment and the sequenceand/or length of the CTO.

It is a striking feature of the present invention that the adjustableT_(m) value of the extended duplex is employed to give a target signalindicative of the presence of the extended duplex by melting theextended duplex in the step (e).

The term used herein “T_(m)” refers to a melting temperature at whichhalf a population of double stranded nucleic acid molecules aredissociated to single-stranded molecules. The T_(m) value is determinedby length and G/C content of nucleotides hybridized. The T_(m) value maybe calculated by conventional methods such as Wallace rule (R. B.Wallace, et al., Nucleic Acids Research, 6:3543-3547(1979)) andnearest-neighbor method (Santa Lucia J. Jr., et al., Biochemistry,35:3555-3562(1996)); Sugimoto N., et al., Nucleic Acids Res.,24:4501-4505(1996)).

According to a preferred embodiment, the T_(m) value refers to actualT_(m) values under reaction conditions actually practiced.

The template-dependent nucleic acid polymerase used in the step (d) mayinclude any nucleic acid polymerases, for example, Klenow fragment of E.coli DNA polymerase I, a thermostable DNA polymerase and bacteriophageT7 DNA polymerase. Preferably, the polymerase is a thermostable DNApolymerase which may be obtained from a variety of bacterial species,including Thermus aquaticus (Taq), Thermus thermophilus (Tth), Thermusflavus, Thermococcus literalis, Thermus antranikianii, Thermuscaldophilus, Thermus chliarophilus, Thermus flavus, Thermus igniterrae,Thermus lacteus, Thermus oshimai, Thermus ruber, Thermus rubens, Thermusscotoductus, Thermus silvanus, Thermus species Z05, Thermus species sps17, Thermus thermophilus, Thermotoga maritima, Thermotoga neapolitana,Thermosipho africanus, Thermococcus litoralis, Thermococcus barossi,Thermococcus gorgonarius, Thermotoga maritima, Thermotoga neapolitana,Thermosiphoafricanus, Pyrococcus furiosus (Pfu), Pyrococcus woesei,Pyrococcus horikoshii, Pyrococcus abyssi, Pyrodictium occultum, Aquifexpyrophilus and Aquifex aeolieus. Most preferably, the template-dependentnucleic acid polymerase is Taq polymerase.

According to a preferred embodiment, the enzyme having the 5′ nucleaseactivity used in the step (b) is identical to the template-dependentnucleic acid polymerase used in the step (d). More preferably, theenzyme having the 5′ nuclease activity used in the step (b), thetemplate-dependent nucleic acid polymerase used for extension of theupstream primer and the template-dependent nucleic acid polymerase usedin the step (d) are identical to one another.

The extended duplex has a label originated from (i) at least one labellinked to the PTO fragment and/or the CTO, (ii) a label incorporatedinto the extended duplex during the extension reaction, (iii) a labelincorporated into the extended duplex during the extension reaction anda label linked to the PTO fragment and/or the CTO, or (iv) anintercalating label.

The presence of the extended duplex can indicate the presence of thetarget nucleic acid sequence because the extended duplex is formed whenthe target nucleic acid sequence is present. For detecting the presenceof the extended duplex in a direct fashion, an extended duplex having alabel providing a detectable signal is formed in the step (d). The labelused on the extended duplex provides a signal change depending onwhether the extended duplex is in a double strand or single strand,finally giving the target signal indicative of the presence of theextended duplex by melting of the extended duplex.

Step (e): Melting of the Extended Duplex

Following the extension reaction, the extended duplex is melted over arange of temperatures to give a target signal indicative of the presenceof the extended duplex

The target signal is provided by (i) at least one label linked to thefragment and/or the CTO, (ii) a label incorporated into the extendedduplex during the extension reaction, (iii) a label incorporated intothe extended duplex during the extension reaction and a label linked tothe fragment and/or the CTO, or (iv) an intercalating label.

The term used herein “target signal” means any signal capable ofindicating the presence of the extended duplex. For example, the targetsignal includes a signal from labels (signal generation orextinguishment), a signal change from labels (signal increase ordecrease), a melting curve, a melting pattern and a melting temperature(or T_(m) value).

According to a preferred embodiment, the target signal is a signalchange from the label on the extended duplex in the melting step. Thesignal change may be obtained by measuring signals at not less than twodifferent temperatures. Alternatively, the target signal is a meltingcurve, a melting pattern and a melting temperature (or T_(m) value)obtained by measuring signals from the label on the extended duplex overa range of temperatures. Preferably, the range of temperatures is arange of temperatures for a melting curve analysis or temperaturesaround the T_(m) value of the extended duplex.

The extended duplex has higher T_(m) value than the hybrid between theuncleaved PTO and the CTO. Therefore, the extended duplex and the hybridexhibit different melting patterns from each other. Such differentmelting patterns permit to discriminate a target signal from non-targetsignals. The different melting pattern or melting temperature generatesthe target signal together with a suitable labeling system.

The melting may be carried out by conventional technologies, including,but not limited to, heating, alkali, formamide, urea and glycoxaltreatment, enzymatic methods (e.g., helicase action), and bindingproteins. For instance, the melting can be achieved by heating attemperature ranging from 80° C. to 105° C. General methods foraccomplishing this treatment are provided by Joseph Sambrook, et al.,Molecular Cloning, A Laboratory Manual, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (200).

The suitable labeling systems used in this invention are various interms of their types, locations and signal generation fashion.

The labeling systems useful in this invention will be described indetail as follows:

(i) Label Linked to the Fragment and/or the CTO

According to a preferred embodiment, the target signal is provided by atleast one label linked to the fragment and/or the CTO. As the extendedduplex is formed between the PTO fragment and CTO, either the label onthe PTO fragment or on the CTO is present on the extended duplex,providing the target signal in the melting step.

The label includes an interactive dual label and a single label.

(i-1) Interactive Dual Label

The interactive label system is a signal generating system in whichenergy is passed non-radioactively between a donor molecule and anacceptor molecule. As a representative of the interactive label system,the FRET (fluorescence resonance energy transfer) label system includesa fluorescent reporter molecule (donor molecule) and a quencher molecule(acceptor molecule). In FRET, the energy donor is fluorescent, but theenergy acceptor may be fluorescent or non-fluorescent. In another formof interactive label systems, the energy donor is non-fluorescent, e.g.,a chromophore, and the energy acceptor is fluorescent. In yet anotherform of interactive label systems, the energy donor is luminescent, e.g.bioluminescent, chemiluminescent, electrochemiluminescent, and theacceptor is fluorescent. The donor molecule and the acceptor moleculemay be described as a reporter molecular and a quencher molecule in thepresent invention, respectively.

Preferably, the signal indicative of the presence of the extended duplex(i.e., the presence of the target nucleic acid sequence) is generated byinteractive label systems, more preferably the FRET label system (i.e.,interactive dual label system).

First Embodiment Intrastrand Interactive-Dual Label

In a first embodiment of an interactive dual label system, the fragmentor the CTO has an interactive dual label comprising a reporter moleculeand a quencher molecule; wherein the melting of the extended duplex inthe step (e) induces change of a signal from the interactive dual labelto give the target signal in the step (e). The first embodiment of theinteractive dual label system is illustrated in FIGS. 2, 6 and 9. Thefirst embodiment is named as an intrastrand interactive-dual label.

First Embodiment in FIG. 2 Intrastrand Interactive-Dual Label

The exemplified embodiment is described with referring to FIG. 2. Thetemplating portion of the CTO has a reporter molecule and a quenchermolecule. The PTO hybridized with the target nucleic acid sequence isdigested to release the fragment and the fragment is hybridized with thecapturing portion of the CTO and extended to form the extended duplex.

When the extended duplex is formed in the step (d), the reportermolecule and the quencher molecule on the CTO are conformationallyseparated to allow the quencher molecule to unquench the signal from thereporter molecule; wherein when the extended duplex is melted in thestep (e), the reporter molecule and the quencher molecule areconformationally adjacent to each other to allow the quencher moleculeto quench the signal from the reporter molecule, such that the targetsignal is given to indicate the presence of the extended duplex in thestep (e).

The expression used herein “the reporter molecule and the quenchermolecule are conformationally adjacent” means that the reporter moleculeand the quencher molecule are three-dimensionally adjacent to each otherby a conformational structure of the fragment or CTO such as random coiland hairpin structure.

The expression used herein “the reporter molecule and the quenchermolecule are conformationally separated” means that the reportermolecule and the quencher molecule are three-dimensionally separated bychange of a conformational structure of the fragment or CTO upon theformation of a double strand.

Preferably, the target signal given in the step (e) includes meltingcurve, a melting pattern or a T_(m) value that is obtained by measuringchange of the fluorescent signal generated in the step (d).

According to a preferred embodiment, the reporter molecule and thequencher molecule may be located at any site on the CTO, so long as thesignal from the reporter molecule is quenched and unquenched dependingon melting of the extended duplex.

According to a preferred embodiment, the reporter molecule and thequencher molecule both are linked to the templating portion or to thecapturing portion of the CTO.

According to a preferred embodiment, the reporter molecule and thequencher molecule are positioned at 5′-end and 3′-end of CTO.

According to a preferred embodiment, one of the reporter molecule andthe quencher molecule on the CTO is located at its 5′-end or at 1-5nucleotides apart from its 5′-end and the other is located to quench andunquench the signal from the reporter molecule depending on conformationof CTO.

According to the preferred embodiment, one of the reporter molecule andthe quencher molecule on the CTO is located at its 3′-end or at 1-5nucleotides apart from its 3′-end and the other is located to quench andunquench the signal from the reporter molecule depending on conformationof CTO.

According to a preferred embodiment, the reporter molecule and thequencher molecule are positioned at no more than 80 nucleotides, morepreferably no more than 60 nucleotides, still more preferably no morethan 30 nucleotides, still much more preferably no more than 25nucleotides apart from each other. According to a preferred embodiment,the reporter molecule and the quencher molecule are separated by atleast 4 nucleotides, more preferably at least 6 nucleotides, still morepreferably at least 10 nucleotides, still much more preferably at least15 nucleotides.

In the present invention, a hybrid between the uncleaved PTO and the CTOmay be formed.

Where the templating portion of the CTO is labeled with an interactivedual label as shown in FIG. 2, a signal change from the label on thehybrid between the uncleaved PTO and the CTO is not induced. Therefore,the hybrid does not provide a non-target signal.

Where the capturing portion of the CTO is labeled with an interactivedual label, the hybrid between the uncleaved PTO and the CTO provides anon-target signal in the melting step. In this case, the difference inT_(m) values of the extended duplex and the hybrid permits todiscriminate the target signal of the extended duplex from thenon-target signal of the hybrid.

First Embodiment in FIG. 6 Intrastrand Interactive-Dual Label

The exemplified embodiment is described with referring to FIG. 6. The5′-tagging portion of the PTO has a reporter molecule and a quenchermolecule. The PTO hybridized with the target nucleic acid sequence isdigested to release the fragment comprising the 5′-tagging portion withthe reporter molecule and the quencher molecule. The fragment ishybridized with the capturing portion of the CTO.

When the extended duplex is formed in the step (d), the reportermolecule and the quencher molecule on the fragment are conformationallyseparated to allow the quencher molecule to unquench the signal from thereporter molecule; wherein when the extended duplex is melted in thestep (e), the reporter molecule and the quencher molecule areconformationally adjacent to each other to allow the quencher moleculeto quench the signal from the reporter molecule, such that the targetsignal is given to indicate the presence of the extended duplex in thestep (e).

According to a preferred embodiment, the reporter molecule and thequencher molecule may be located at any site on the fragment, so long asthe signal from the reporter molecule is quenched and unquencheddepending on melting of the extended duplex.

According to a preferred embodiment, one of the reporter molecule andthe quencher molecule on the fragment is located at its 5′-end or at 1-5nucleotides apart from its 5′-end and the other is located to quench andunquench the signal from the reporter molecule depending on conformationof the fragment.

According to a preferred embodiment, the reporter molecule and thequencher molecule are positioned at no more than 50 nucleotides, morepreferably no more than 40 nucleotides, still more preferably no morethan 30 nucleotides, still much more preferably no more than 20nucleotides apart from each other. According to a preferred embodiment,the reporter molecule and the quencher molecule are separated by atleast 4 nucleotides, more preferably at least 6 nucleotides, still morepreferably at least 10 nucleotides, still much more preferably at least15 nucleotides.

As represented in FIG. 6, the hybrid between the uncleaved PTO and theCTO provides a non-target signal in the melting step. In this case, thedifference in T_(m) values of the extended duplex and the hybrid permitsto discriminate the target signal of the extended duplex from thenon-target signal of the hybrid.

Second Embodiment Interstrand Interactive-Dual Label

In the second embodiment of the interactive label system, the fragmenthas one of an interactive dual label comprising a reporter molecule anda quencher molecule and the CTO has the other of the interactive duallabel; wherein the melting of the extended duplex in the step (e)induces change of a signal from the interactive dual label to give thetarget signal in the step (e).

The exemplified embodiment is described with referring to FIG. 8.

When the extended duplex is formed in the step (d), the signal from thereporter molecule linked to the CTO is quenched by the quencher moleculelinked to the PTO. When the extended duplex is melted in the step (e),the reporter molecule and the quencher molecule are separated to allowthe quencher molecule to unquench the signal from the reporter molecule,such that the target signal is given to indicate the presence of theextended duplex in the step (e).

Preferably, the target signal given in the step (e) includes a meltingcurve, a melting pattern or a T_(m) value that is obtained by measuringchange of the fluorescent signal from the interactive dual label.

The reporter molecule and the quencher molecule may be located at anysite of the PTO fragment and the CTO, so long as the signal from thereporter molecule is quenched by the quencher molecule in the extendedduplex.

According to a preferred embodiment, the reporter molecule or thequencher molecule on the PTO fragment is located at the 5′-end of the5′-tagging portion.

According to a preferred embodiment, the reporter molecule or thequencher molecule on the CTO is located at its 3′-end.

As represented in FIG. 8, the hybrid between the uncleaved PTO and theCTO provides a non-target signal in the melting step. In this case, thedifference in T_(m) values of the extended duplex and the hybrid permitsto discriminate the target signal of the extended duplex from thenon-target signal of the hybrid.

The reporter molecule and the quencher molecule useful in the presentinvention may include any molecules known in the art, Examples of thoseare: Cy2™ (506), YO-PRO™-1 (509), YOYO™-1 (509), Calcein (517), FITC(518), FluorX™ (519), Alexa™ (520), Rhodamine 110 (520), Oregon Green™500 (522), Oregon Green™ 488 (524), RiboGreen™ (525), Rhodamine Green™(527), Rhodamine 123 (529), Magnesium Green™ (531), Calcium Green™(533), TO-PRO™-1 (533), TOTO1 (533), JOE (548), BODIPY530/550 (550), Dil(565), BODIPY TMR (568), BODIPY558/568 (568), BODIPY564/570 (570), Cy3™(570), Alexa™ 546 (570), TRITC (572), Magnesium Orange™ (575),Phyeoerythrin R&B (575), Rhodamine Phalloidin (575), Calcium Orange™(576), Pyronin Y (580), Rhodamine B (580), TAMRA (582), Rhodamine Red™(590), Cy3.5™ (596), ROX (608), Calcium Crimson™ (615), Alexa™ 594(615), Texas Red® (615), Nile Red (628), YO-PRO™-3 (631), YOYO™-3 (631),R-phycocyanin (642), C-Phycocyanin (648), TO-PRO™-3 (660), TOTO3 (660),DiD DilC(5) (665), CY5™ (670), Thiadicarbocyanine (671), Cy5.5 (694),HEX (556), TET (536), Biosearch Blue™ (447), CAL Fluor® dyes such as CALFluor Gold 540 (544), CAL Fluor Orange 560 (559), CAL Fluor Red 590(591), CAL Fluor Red 610 (610), CAL Fluor Red 635 (637), FAM (520),Fluorescein (520), Fluorescein-C3 (520), Pulsar 650 (566), Quasar 570(667), Quasar 670 (705) and Quasar 705 (610), The numeric in parenthesisis a maximum emission wavelength in nanometer. Preferably, the reportermolecule and the quencher molecule include JOE, PAM, TAMRA, ROX andfluorescein-based label.

Suitable pairs of reporter-quencher are disclosed in a variety ofpublications as follows: Pesce et al., editors, FluorescenceSpectroscopy (Marcel Dekker, New York, 1971); White et al., FluorescenceAnalysis: A Practical Approach (Marcel Dekker, New York, 1970); Berlman,Handbook of Fluorescence Spectra of Aromatic Molecules, 2^(nd) Edition(Academic Press, New York, 1971); Griffiths, Color AND Constitution ofOrganic Molecules (Academic Press, New York, 1976); Bishop, editor,Indicators (Pergamon Press, Oxford, 1972); Haugland, Handbook ofFluorescent Probes and Research Chemicals (Molecular Probes, Eugene,1992); Pringsheim, Fluorescence and Phosphorescence (IntersciencePublishers, New York, 1949); Haugland, R. P., Handbook of FluorescentProbes and Research Chemicals, 6^(th) Edition (Molecular Probes, Eugene,Oreg., 1996) U.S. Pat. Nos. 3,996,345 and 4,351,760.

It is noteworthy that a non-fluorescent black quencher molecule capableof quenching a fluorescence of a wide range of wavelengths or a specificwavelength may be used in the present invention. Examples of those areBHQ and DABCYL.

In the FRET label adopted to the CTO, the reporter encompasses a donorof FRET and the quencher encompasses the other partner (acceptor) ofFRET. For example, a fluorescein dye is used as the reporter and arhodamine dye as the quencher.

(i-2) Single Label

The present invention is also excellently executed using single labelsystems for providing signals indicating the presence of target nucleicacid sequences.

According to a preferred embodiment, the fragment or the CTO has asingle label, and the melting of the extended duplex in the step (e)induces change of a signal from the single label to give the targetsignal in the step (e).

First Embodiment in FIG. 3 Single Label System

The exemplified embodiment is described with referring to FIG. 3. Thetemplating portion of the CTO has a single fluorescent label. The PTOhybridized with the target nucleic acid sequence is digested to releasethe fragment. The fragment is hybridized with the capturing portion ofthe CTO and extended to form the extended duplex. By the formation ofthe extended duplex, the fluorescent intensity from the singlefluorescent label becomes increased. When the extended duplex is meltedin the step (e), the fluorescent intensity from the single fluorescentlabel becomes decreased, such that the target signal is given toindicate the presence of the extended duplex in the step (e).

According to a preferred embodiment, the single label may be located atany site on the CTO, so long as the signal level from the single labelis changed depending on melting of the extended duplex.

According to a preferred embodiment, the single label is linked to thetemplating portion or to the capturing portion of the CTO.

Where the templating portion of the CTO is labeled with a single labelas shown in FIG. 3, a signal change from the label on the hybrid betweenthe uncleaved PTO and the CTO is not induced. Therefore, the hybrid doesnot provide a non-target signal.

Where the capturing portion of the CTO is labeled with a single label,the in hybrid between the uncleaved PTO and the CTO provides anon-target signal in the melting step. In this case, the difference inT_(m) values of the extended duplex and the hybrid permits todiscriminate the target signal of the extended duplex from thenon-target signal of the hybrid.

Second Embodiment in FIG. 7 Single Label System

The exemplified embodiment is described with reference to FIG. 7. The5′-tagging portion of the PTO has a single fluorescent label. The PTOhybridized with the target nucleic acid sequence is digested to releasethe fragment comprising the 5′-tagging portion with the singlefluorescent label. By the hybridization, the signal intensity from thesingle fluorescent label on the 5′-tagging portion is increased. Whenthe extended duplex is melted in the step (e), the signal intensity fromthe single fluorescent label becomes decreased, such that the targetsignal is given to indicate the presence of the extended duplex in thestep (e).

According to a preferred embodiment, the single label may be located atany site on the mu fragment, so long as the signal level from the singlelabel is changed depending on melting of the extended duplex.

As represented in FIG. 7, the hybrid between the uncleaved PTO and theCTO provides a non-target signal in the melting step. In this case, thedifference in T_(m) values of the extended duplex and the hybrid permitsto discriminate the target signal of the extended duplex from thenon-target signal of the hybrid.

The single label used herein has to be capable of providing a differentsignal depending on its presence on a double strand or single strand.The single label includes a fluorescent label, a luminescent label, achemiluminescent label, an electrochemical label and a metal label.Preferably, the single label includes a fluorescent label.

The types and preferable binding sites of single fluorescent labels usedin this invention are disclosed U.S. Pat. Nos. 7,537,886 and 7,348,141,the teachings of which are incorporated herein by reference in theirentity. Preferably, the single fluorescent label includes JOE, FAM,TAMRA, ROX and fluorescein-based label. The labeled nucleotide residueis preferably positioned at internal nucleotide residue within theoligonucleotide rather than at the 5′-end or the 3′-end.

The single fluorescent label useful in the present invention may bedescribed with reference to descriptions for reporter and quenchermolecules as indicated above.

In particular, where the present invention on a solid phase is performedusing a single label, it can utilize a general fluorescent label anddoes not require a specific fluorescent label capable of providing afluorescent signal with different intensities depending on its presenceon double strand or single strand. The target signal provided on thesolid substrate is measured. The embodiment of the single label systemwith immobilized CTO is illustrated in FIG. 12.

When the CTO immobilized onto a solid substrate is used, chemical labels(e.g., biotin) or enzymatic labels (e.g. alkaline phosphatase,peroxidase, β-galactosidase and β-glucosidase) may be used.

In the labeling system using “label linked to the fragment and/or theCTO”, the labels may be positioned to the extent that when a hybridbetween an uncleaved PTO and the CTO is formed, the hybrid does not givea non-target signal in the step (e). Alternatively, the labels may bepositioned to the extent that when a hybrid between an uncleaved PTO andthe CTO is formed, the hybrid gives a non-target signal in the step (e);wherein the T_(m) value of the extended duplex is higher than that ofthe hybrid between the uncleaved PTO and the CTO.

Particularly, where the labels are positioned to the extent that ahybrid between an uncleaved PTO and the CTO does not give a non-targetsignal, the range including T_(m) value of the hybrid can be utilized toselect T_(m) value of the extended duplex for detecting a target nucleicacid sequence.

(ii) Label Incorporated into the Extended Duplex

The present invention may employ a label incorporated into the extendedduplex during the extension reaction for providing the target signalindicative of the presence of the extended duplex.

Although the PTO fragment or CTO has no label, a label incorporated intothe extended duplex during the extension reaction is successfully usedto allow the extended duplex to be labeled. FIGS. 10 and 11 illustratean embodiment in which a single-labeled nucleotide is incorporated intothe extended duplex during the extension reaction (see C and D of FIGS.10 and 11). This embodiment is also applicable to other embodimentsusing a melting analysis.

According to a preferred embodiment, the target signal is provided by asingle label incorporated into the extended duplex during the extensionreaction; wherein the incorporated single label is linked to anucleotide incorporated during the extension reaction; wherein themelting of the extended duplex in the step (e) induces change of asignal from the single label to give the target signal in the step (e).

The exemplified embodiment is described with reference to FIG. 10. ThePTO hybridized with the target nucleic acid sequence is digested torelease the fragment. The fragment is hybridized with the capturingportion of the CTO immobilized on a solid substrate and extended in thepresence of nucleotides labeled with the single fluorescent label toform the extended duplex. The fluorescent signal from the extendedduplex may be detected on spot of the solid substrate with immobilizedCTO. When the extended duplex is melted, a strand having a fluorescentlabel is released and the fluorescent signal is no longer detected onthe spot (not shown in FIG. 10). Therefore, a signal change can beprovided on the spot by melting of the extended duplex. In this regard,the target signal is given to indicate the presence of the extendedduplex in the step (e).

The target signal given in the step (e) includes a melting curve, amelting pattern or a T_(m) value that is obtained by measuring change ofthe fluorescent intensity on the CTO-immobilized spot.

According to a preferred embodiment, a nucleotide incorporated duringthe extension reaction has a first non-natural base and the CTO has anucleotide having a second non-natural base with a specific bindingaffinity to the first non-natural base, as illustrated in FIG. 11. Thenucleotide having the second non-natural base is preferably located atany site on the templating portion of the CTO.

The term used herein “non-natural base” refers to derivatives of naturalbases such as adenine (A), guanine (C), thymine (T), cytosine (C) anduracil (U), which are capable of forming hydrogen-bonding base pairs.The term used herein “non-natural base” includes bases having differentbase pairing patterns from natural bases as mother compounds, asdescribed, for example, in U.S. Pat. Nos. 5,432,272, 5,965,364,6,001,983, and 6,037,120. The base pairing between non-natural basesinvolves two or three hydrogen bonds as natural bases. The base pairingbetween non-natural bases is also formed in a specific manner.

Specific examples of non-natural bases include the following bases inbase pair combinations: iso-C/iso-G, iso-dC/iso-dG, K/X, H/J, and M/N(see U.S. Pat. No. 7,422,850).

The exemplified embodiment is described with reference to FIG. 11. Thefragment is hybridized with the CTO with a nucleotide having a secondnon-natural base (e.g., iso-dC) with a specific binding affinity to afirst non-natural base (e.g., iso-dG). The extension is carried out inthe presence of a nucleotide having the first non natural base labeledwith a single fluorescent label, forming the extended duplex. In theextension reaction, the nucleotide having the first non-natural base isincorporated at an opposition site to the nucleotide having the secondnon-natural base.

The fluorescent signal from the extended duplex may be detected on spotof a solid substrate with immobilized CTO. When the extended duplex ismelted, a strand having a fluorescent label is released and thefluorescent signal is no longer detected on the spot (not shown in FIG.11). Therefore, a signal change can be provided on the spot by meltingof the extended duplex. In this regard, the target signal is given toindicate the presence of the extended duplex in the step (e).

Where the label incorporated into the extended duplex during theextension reaction is employed, the label is not incorporated into thehybrid between the uncleaved PTO and the CTO because the hybrid is notextended. Therefore, the hybrid does not provide a non-target signal.

The types and characteristics of the single labels used may be describedwith reference to descriptions for the labeling system using “labellinked to the fragment and/or the CTO” as indicated hereinabove.

(iii) Label Incorporated into the Extended Duplex and Label Linked tothe Fragment or the CTO

The present invention may employ a labeling system using cooperation ofa label incorporated into the extended duplex during the extensionreaction and a label linked to the fragment and/or the CTO, asillustrated in FIGS. 4 and 5.

According to a preferred embodiment, the target signal is provided by alabel incorporated into the extended duplex during the extensionreaction and a label linked to the fragment and/or the CTO, and theincorporated label is linked to a nucleotide incorporated during theextension reaction; wherein the two labels are an interactive dual labelof a reporter molecule and a quencher molecule; wherein the melting ofthe extended duplex in the step (e) induces change of a signal from theinteractive dual label to give the target signal in the step (e).

More preferably, the nucleotide incorporated during the extensionreaction has a first non-natural base and the CTO has a nucleotidehaving a second non-natural base with a specific binding affinity to thefirst non-natural.

The exemplified embodiment is described with reference to FIG. 4. Thefragment is hybridized with the CTO comprising a reporter or quenchermolecule and a nucleotide having a second non-natural base (e.g.,iso-dC) which is a specific binding affinity to a first non-natural base(e.g., iso-dC). The extension is carried out in the presence of anucleotide having the first non-natural base labeled with a quencher orreporter molecule, forming the extended duplex in which the signal fromthe reporter molecule is quenched by the quencher molecule. In theextension reaction, the nucleotide having the first non-natural base isincorporated at an opposition site to the nucleotide having the secondnon-natural base.

When the extended duplex is melted in the step (e), the reportermolecule and the quencher molecule are separated to allow the quenchermolecule to unquench the signal from the reporter molecule, such thatthe target signal is given to indicate the presence of the extendedduplex in the step (e).

Preferably, the target signal given in the step (e) includes a meltingcurve, a melting pattern or a T_(m) value that is obtained by measuringchange of the signal from the interactive dual label.

The site of the label on the CTO and the incorporation site of the labelincorporated are determined to the extent that the two labels are actedas an interactive dual label for inducing signal change in the meltingstep.

Still more preferably, the templating portion of the CTO has a reporteror quencher molecule and a nucleotide having a second non-natural base.The extension reaction in the step (d) is performed in the presence of anucleotide having a quencher or reporter molecule and a firstnon-natural base with a specific binding affinity to the secondnon-natural base in the CTO. The two non-natural bases in the extendedduplex in the step (d) form a base-pairing to quench a signal from thereporter molecule by the quencher molecule and to induce change of asignal, whereby the target signal is provided. Alternatively, thefragment has a reporter or quencher molecule and the templating portionof the CTO has a nucleotide having a second non-natural base. Theextension reaction in the step (d) is performed in the presence of anucleotide having a quencher or reporter molecule and a firstnon-natural base with a specific binding affinity to the secondnon-natural base in the CTO. The two non-natural bases in the extendedduplex in the step (d) form a base-pairing to induce change a signalfrom the reporter molecule by quenching, whereby the target signal isprovided.

Another exemplified embodiment is described with reference to FIG. 5. Inthis embodiment, the fragment having a reporter or quencher molecule ishybridized with the CTO comprising a nucleotide having a secondnon-natural base (e.g. iso-dC) which is a specific binding affinity to afirst non-natural base (e.g., iso-dG). The extension is carried out inthe presence of a nucleotide having the first non-natural base labeledwith a quencher or reporter molecule, forming the extended duplex inwhich the signal from the reporter molecule is quenched by the quenchermolecule. In the extension reaction, the nucleotide having the firstnon-natural base is incorporated at an opposition site to the nucleotidehaving the second non-natural base.

When the extended duplex is formed in the step (d), the reportermolecule and the quencher molecule are conformationally separated toallow the quencher molecule to unquench the signal from the reportermolecule; wherein when the extended duplex is melted in the step (e),the reporter molecule and the quencher molecule are conformationallyadjacent to each other to allow the quencher molecule to quench thesignal from the reporter molecule, such that the target signal is givento indicate the presence of the extended duplex in the step (e).

Preferably, the target signal given in the step (e) includes a meltingcurve, a melting pattern or a T_(m) value that is obtained by measuringchange of the signal from the interactive dual label.

The site of the label on the PTO and the incorporation site of the labelincorporated are determined to the extent that the two labels are actedas an interactive dual label for inducing signal change in the meltingstep.

Where the label incorporated into the extended duplex during theextension reaction is employed, the label is not incorporated into thehybrid between the uncleaved PTO and the CTO because the hybrid is notextended. Therefore, the hybrid does not provide a non-target signal inthe melting step.

(iv) Intercalating Label

The present invention may employ an intercalating label for providingthe target signal indicative of the presence of the extended duplex. Theintercalating label is more useful on a solid phase reaction usingimmobilized CTOs because double-stranded nucleic acid molecules presentin samples can generate signals.

Exemplified intercalating dyes useful in this invention include SYBR™Green I, PO-PRO™-1, BO-PRO™-1, SYTO™ 43, SYTO™44, SYTO™45, SYTOX™Blue,POPO™-1, POPO™3, BOBO™-1, BOBO™-3, LO-PRO™-1, JO-PRO™-1, YO-PRO™1,TO-PRO™1, SYTO™11, SYTO™13, SYTO™15, SYTO™16, SYTO™20, SYTO™23, TOTO™-3,YOYO™3, GelStar™ and thiazole orange. The intercalating dyes intercalatespecifically into double-stranded nucleic acid molecules to generatesignals.

FIG. 13 illustrates an embodiment in which intercalating dyesintercalate between base-pairs of the extended duplex (C and D in FIG.13). The embodiment is also applicable to another embodiment using amelting analysis.

The exemplified embodiment is described with reference to FIG. 13. Thefragment is hybridized with the capturing portion of the CTO immobilizedon a solid substrate. The extension is carried out in the presence of anintercalating dye (e.g., SYBR™ Green) and produces the extended duplexwith intercalating dyes. The fluorescent signal from the extended duplexon spot of the solid substrate with immobilized CTO may be detectedusing intercalating fluorescent dyes. When the extended duplex ismelted, intercalating fluorescent dyes are released and the fluorescentsignal is no longer detected on the spot (not shown in FIG. 13). In thisregard, the target signal is given to indicate the presence of theextended duplex in the step (e).

The hybrid between the uncleaved PTO and the CTO provides a non-targetsignal in the melting step. In this ease, the difference in T_(m) valuesof the extended duplex and the hybrid permits to discriminate the targetsignal of the extended duplex from the non-target signal of the hybrid(not shown in FIG. 13).

Preferably, the target signal given in the step (e) includes a meltingcurve, a melting pattern or a T_(m) value that is obtained by measuringchange of the fluorescent signal generated in the step (d).

Step (f): Detection of Target Signal

Finally, the extended duplex is detected by measuring the target signalgiven in the step (e), whereby the presence of the extended duplexindicates the presence of the target nucleic acid sequence.

The detection may be carried out in various manners depending on thetypes of the target signal.

According to a preferred embodiment, the detection of the target signalis carried out by a melting analysis.

The term used herein “melting analysis” means a method in which a targetsignal indicative of the presence of the extended duplex is obtained bymelting of the extended duplex, including a method to measure signals attwo different temperatures, melting curve analysis, melting patternanalysis and melting peak analysis. Preferably, the melting analysis isa melting curve analysis.

According to a preferred embodiment, the melting of the step (e) isfollowed by hybridization to give the target signal indicative of thepresence of the extended duplex. In that case, the presence of theextended duplex is detected by hybridization curve analysis.

The melting curve or hybridization curve may be obtained by conventionaltechnologies, for example, as described in U.S. Pat. Nos. 6,174,670 and5,789,167, Drobyshev et al, Gene 188: 45(1997); Kochinsky and MirzabekovHuman Mutation 19:343(2002); Livehits et al J. Biomol. Structure Dynam.11:783(1994); and Howell et al Nature Biotechnology 17:87(1999). Forexample, a melting curve or hybridization curve may consist of a graphicplot or display of the variation of the output signal with the parameterof hybridization stringency. Output signal may be plotted directlyagainst the hybridization parameter. Typically, a melting curve orhybridization curve will have the output signal, for examplefluorescence, which indicates the degree of duplex structure (i.e. theextent of hybridization), plotted on the Y-axis and the hybridizationparameter on the X axis.

The PTO and CTO may be comprised of naturally occurring dNMPs.Alternatively, the PTO and CTO may be comprised of modified nucleotideor non-natural nucleotide such as PNA (peptide nucleic acid, see PCTPublication No. WO 92/20702) and LNA (locked nucleic acid, see PCTPublication Nos. WO 98/22489, WO 98/39352 and WO 99/14226). The PTO andCTO may comprise universal bases such as deoxyinosine, inosine,1-(2′-deoxy-beta-D-ribofuranosyl)-3-nitropyrrole and 5-nitroindole. Theterm “universal base” refers to one capable of forming base pairs witheach of the natural DNA/RNA bases with little discrimination betweenthem.

As described above, the PTO may be cleaved at a site located in a3′-direction apart from the 3′-end of the 5′-tagging portion of the PTO.The cleavage site may be located at the 5′-end part of the 3′-targetingportion of the PTO. Where the PTO fragment comprises the 5′-end part ofthe 3′-targeting portion of the PTO, a site of the CTO hybridized withthe 5′-end part of the 3′-targeting portion may comprise a universalbase, degenerate sequence or their combination. For instance, if the PTOis cleaved at a site located one nucleotide in a 3′-direction apart fromthe 3′-end of the 5′-tagging portion of the PTO, it is advantageous thatthe 5′-end part of the capturing portion of the CTO comprises auniversal base for hybridization with the nucleotide. If the PTO iscleaved at a site located two nucleotides in a 3′-direction apart fromthe 3′-end of the 5′-tagging portion of the PTO, it is advantageous thatthe 5′-end of the capturing portion of the CTO comprises a degeneratesequence and its 3′-direction-adjacent nucleotide comprises a universalbase. As such, where the cleavage of the PTO occurs at various sites ofthe 5′-end part of the 3′-targeting portion, the utilization ofuniversal bases and degenerate sequences in the CTO is useful. Inaddition, where the PTOs having the same 5′-tagging portion are used forscreening multiple target nucleic acid sequences under upstream primerextension-dependent cleavage induction, the PTO fragments havingdifferent 5′-end parts of the 3′-targeting portion may be generated. Insuch cases, universal bases and degenerate sequences are usefullyemployed in the CTO. The strategies using universal bases and degeneratesequences in the CTO ensure to use one type or minimal types of the CTOfor screening multiple target nucleic acid sequences.

According to a preferred embodiment, the method further comprisesrepeating the steps (a)-(b), (a)-(d) or (a)-(f) with denaturationbetween repeating cycles preferably, with a downstream primer. Thisrepetition permits to amplify the target nucleic acid sequence and/orthe target signal.

According to a preferred embodiment, the steps (a)-(f) are performed ina reaction vessel or in separate reaction vessels. For example, thesteps (a)-(b), (c)-(d) or (e)-(f) may be performed in separate reactionvessels.

According to a preferred embodiment, the steps (a)-(b) and (c)-(f) maybe simultaneously or separately even in a reaction vessel depending onreaction conditions (particularly, temperature).

The present invention does not require that target nucleic acidsequences to be detected and/or amplified have any particular sequenceor length, including any DNA (gDNA and cDNA) and RNA molecules.

Where a mRNA is employed as starting material, a reverse transcriptionstep is necessary prior to performing annealing step, details of whichare found in Joseph Sambrook, et al., Molecular Cloning, A LaboratoryManual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.(2001); and Noonan, K. F. et al., Nucleic Acids Res, 16:10366 (1938).For reverse transcription, a random hexamer or an oligonucleotide dTprimer hybridizable to mRNA can be used.

The target nucleic acid sequences which may be detected and/or amplifiedinclude any naturally occurring prokaryotic, eukaryotic (for example,protozoans and parasites, fungi, yeast, higher plants, lower and higheranimals, including mammals and humans) or viral (for example, Herpesviruses, HIV, influenza virus, Epstein-Barr virus, hepatitis virus,polio virus, etc.) or viroid nucleic acid.

The present invention is also useful in detection of a nucleotidevariation. Preferably, the target nucleic acid sequence comprises anucleotide variation. The term “nucleotide variation” used herein refersto any single or multiple nucleotide substitutions, deletions orinsertions in a DNA sequence at a particular location among contiguousDNA segments that are otherwise similar in sequence. Such contiguous DNAsegments include a gene or any other portion of a chromosome. Thesenucleotide variations may be mutant or polymorphic allele variations.For example, the nucleotide variation detected in the present inventionincludes SNP (single nucleotide polymorphism), mutation, deletion,insertion, substitution and translocation. Exemplified nucleotidevariation includes numerous variations in a human genome (e.g.,variations in the MTHFR (methylenetetrahydrofolate reductase) gene),variations involved in drug resistance of pathogens andtumorigenesis-causing variations.

In the present invention for detection of a nucleotide variation in atarget nucleic acid sequence, where primers or probes used have acomplementary sequence to the nucleotide variation in the target nucleicacid sequence, the target nucleic acid sequence containing thenucleotide variation is described herein as a matching template. Whereprimers or probes used have a non-complementary sequence to thenucleotide variation in the target nucleic acid sequence, the targetnucleic acid sequence containing the nucleotide variation is describedherein as a mismatching template.

For detection of nucleotide variations, the 3′-end of the upstreamprimer may be designed to be opposite to a site of a nucleotidevariation in a target nucleic acid sequence. According to a preferredembodiment, the 3′-end of the upstream primer has a complementarysequence to the nucleotide variation in a target nucleic acid sequence.The 3′-end of the upstream primer having a complementary sequence to thenucleotide variation in the target nucleic acid sequence is annealed tothe matching template and extended to induce cleavage of the PTO. Theresultant PTO fragment is hybridized with the CTO to provide the targetsignal. In contrast, where the 3′-end of the upstream primer ismismatched to a nucleotide variation in a mismatching template, it isnot extended under conditions that annealing of the 3′-end of primers isessential for extension even when the upstream primer is hybridized withthe mismatching template, thereby resulting in no generation of thetarget signal.

Alternatively, it is possible to use PTO cleavage depending on thehybridization of PTO having a complementary sequence to a nucleotidevariation in a target nucleic acid sequence. For example, undercontrolled conditions, a PTO having a complementary sequence to thenucleotide variation in the target nucleic acid sequence is hybridizedwith the matching template and then cleaved. The resultant PTO fragmentis hybridized with the CTO to provide the target signal. While, underthe controlled conditions, the PTO is not hybridized with a mismatchingtemplate having non-complementary sequence in the nucleotide variationposition and not cleaved. Preferably, in this case, the complementarysequence to the nucleotide variation in the PTO is positioned at itsmiddle of the 3′-targeting portion of the PTO.

Alternatively, it is preferable that the 5′-end part of the 3′-targetingportion of the PTO is positioned to a nucleotide variation in a targetnucleic acid sequence for the detection of the nucleotide variation andthe 5′-end part of the 3′-targeting portion of the PTO has acomplementary sequence to the nucleotide variation in a target nucleicacid sequence.

In an embodiment for the detection of a single nucleotide variation, the5′-end of the 3′-targeting portion of the PTO has a complementarysequence to the single nucleotide variation in a target nucleic acidsequence. As described above, the cleavage of the PTO hybridized with amatching template may be induced at a site immediately adjacent in a3′-direction to the 5′-end of the 3′-targeting portion of the PTO, forexample, under upstream primer extension-dependent cleavage induction.The 3′-end of the PTO fragment has the complementary nucleotide to thesingle nucleotide variation. The PTO fragment is hybridized with a CTOhaving a capturing portion comprising a sequence corresponding to thenucleotide variation and then extended to form the extended duplex,providing the target signal. If the same PTO is hybridized with amismatching template having the identical sequence to the matchingtemplate except for the single nucleotide variation, the cleavage of thePTO may occur at a site two nucleotides apart in a 3′-direction from the5′-end of the 3′-targeting portion of the PTO. The 3′-end of the PTOfragment has the further cleaved nucleotide than the complementarynucleotide to the single nucleotide variation. Where the site of the CTOhybridized with the additional-cleaved nucleotide is designed to have anon-complementary sequence to the further cleaved nucleotide, the 3′-endof the PTO fragment is not hybridized with the CTO, resulting in noextension of the PTO fragment in a controlled condition. Even if the PTOfragment is extended to form the extended duplex, the duplex has adifferent T_(m) value from the duplex derived from hybridization betweenthe PTO and the mismatching template.

According to a preferred embodiment, a cleavage site of the PTO having acomplementary sequence to the nucleotide variation at its 5′-end part ofthe 3′-targeting portion is different depending on hybridization with amatching template or with a mismatching template, such that the PTOfragment released from either hybridization event has different sequencepreferably, in its 3′-end part, more preferably, in its 3′-end.

According to a preferred embodiment, the selection of the nucleotidesequence of CTO in consideration of the difference in 3′-end parts ofthe PTO fragments allows to discriminate the matching template from themismatching template.

According to a preferred embodiment, the target nucleic acid sequenceused in the present invention is a pre-amplified nucleic acid sequence.The utilization of the pre-amplified nucleic acid sequence permits tosignificantly increase the sensitivity and specificity of targetdetection of the present invention.

According to a preferred embodiment, the method is performed in thepresence of a downstream primer.

The advantages of the present invention may be highlighted in thesimultaneous (multiplex) detection of at least two target nucleic acidsequences.

According to a preferred embodiment, the method is performed to detectat least two types (more preferably, at least three types, still morepreferably at least five types) of target nucleic acid sequences.

According to a preferred embodiment, the method is performed to detectat least two types (more preferably, at least three types, still morepreferably at least five types) of target nucleic acid sequences;wherein the upstream oligonucleotide comprises at least two types (morepreferably at least three types, still more preferably at least fivetypes) of oligonucleotides, the PTO comprises at least two types (morepreferably at least three types, still more preferably at least fivetypes) of the PTOs and the CTO comprises at least one type (preferablyat least two types, more preferably at least three types, still morepreferably at least five types) of the CTO; wherein when at least twotypes of the target nucleic acid sequences are present, the methodprovides at least two types of the target signals corresponding to theat least two types of the target nucleic acid sequences.

The 5′-tagging portions of the at least two PTOs may have an identicalsequence to each other. For instance, where the present invention iscarried out for screening target nucleic acid sequences, the 5′-taggingportions of PTOs may have the identical sequence.

Furthermore, a single type of the CTO may used for detection of aplurality of target nucleic acid sequences. For example, where the PTOshaving an identical sequence in their 5′-tagging portions are employedfor screening target nucleic acid sequences, a single type of the CTOmay used.

According to a preferred embodiment, the extended duplexes correspondingto the at least two types of the target nucleic acid sequences havedifferent T_(m) values from each other.

According to a preferred embodiment, the at least two types of thetarget signals corresponding to the at least two types of the targetnucleic acid sequences are provided from different types of labels fromeach other.

According to a preferred embodiment, the at least two types of thetarget signals corresponding to the at least two types of the targetnucleic acid sequences are provided from the same type of labels.

According to a preferred embodiment, the at least two type of the targetsignals corresponding to the at least two types of the target nucleicacid sequences are provided from the same type of labels; wherein theextended duplexes corresponding to the at least two types of the targetnucleic acid sequences have different T_(m) values from each other.

The term used herein “different types of labels” refers to labels withdifferent characteristics of detectable signals. For example, FAM andTAMRA as fluorescent reporter labels are considered as different typesof labels because their excitation and emission wavelengths aredifferent from each other.

Where the present invention is performed to simultaneously detect atleast two types of the target nucleic acid sequences by melting curveanalysis and the extended duplexes corresponding to the at least twotypes of the target nucleic acid sequences have different T_(m) valuesfrom each other, it is possible to detect at least two types of thetarget nucleic acid sequences even using a single type of a label (e.g.FAM).

Target Detection Using immobilized CTO on a Solid Phase

The prominent advantage of the present invention is to be effective indetection of target nucleic acid sequences even on a solid phase such asmicroarray.

According to a preferred embodiment, the present invention is performedon the solid phase and the CTO is immobilized through its 5′-end or3′-end onto a solid substrate. In solid phase, the target signalprovided on the solid substrate is measured.

Where the immobilized CTO is used, the melting analysis using labelingsystems as described above is applicable to the solid phase reaction ofthe present invention.

According to a preferred embodiment, the target signal is provided by asingle label linked to the fragment or by a single label incorporatedinto the extended duplex during the extension reaction. In particular,where the present invention on a solid phase is performed using a singlelabel, it can utilize a general fluorescent label and does not require aspecific fluorescent label capable of providing a fluorescent signalwith different intensities depending on its presence on double strand orsingle strand.

When the CTO immobilized onto a solid substrate is used, chemical labels(e.g. biotin) or enzymatic labels (e.g. alkaline phosphatase,peroxidase, β-galactosidase and β-glucosidase) may be used.

For the solid phase reaction, the CTO is immobilized directly orindirectly (preferably indirectly) through its 5′-end or 3′-end(preferably the 3′-end) onto the surface of the solid substrate.Furthermore, the CTO may be immobilized on the surface of the solidsubstrate in a covalent or non-covalent manner. Where the immobilizedCTOs are immobilized indirectly onto the surface of the solid substrate,suitable linkers are used. The linkers useful in this invention mayinclude any linkers utilized for probe immobilization on the surface ofthe solid substrate. For example, alkyl or aryl compounds with aminefunctionality, or alkyl or aryl compounds with thiol functionality serveas linkers for CTO immobilization. In addition, poly (T) tail or poly(A) tail may serve as linkers.

According to a preferred embodiment, the solid substrate used in thepresent invention is a microarray. The microarray to provide a reactionenvironment in this invention may include any those known to one ofskill in the art. All processes of the present invention, i.e.,hybridization to target nucleic acid sequences, cleavage, extension,melting and fluorescence detection, are carried out on the microarray.The immobilized CTOs on the microarray serve as hybridizable arrayelements. The solid substrate to fabricate microarray includes, but notlimited to, metals (e.g., gold, alloy of gold and copper, aluminum),metal oxide, glass, ceramic, quartz, silicon, semiconductor, Si/SiO₂wafer, germanium, gallium arsenide, carbon, carbon nanotube, polymers(e.g., polystyrene, polyethylene, polypropylene and polyacrylamide),sepharose, agarose and colloids. A plurality of immobilized CTOs in thisinvention may be immobilized on an addressable region or two or moreaddressable regions on a solid substrate that may comprise 2-1,000,000addressable regions. Immobilized CTOs may be fabricated to produce arrayor arrays for a given application by conventional fabricationtechnologies such as photolithography, ink-jetting, mechanicalmicrospotting, and derivatives thereof.

The present invention performed on the solid phase can detectsimultaneously a plurality of target nucleic acid sequences even using asingle type of a label because the labels on the CTOs immobilized arephysically separated. In this regard, the number of target nucleic acidsequences to be detected by the present invention on the solid phase isnot limited.

II. Preferable Embodiment with Amplification of a Target Nucleic AcidSequence

Preferably, the present invention is carried out simultaneously withamplification of a target nucleic acid sequence using a primer paircomposed of an upstream primer and a downstream primer capable ofsynthesizing the target nucleic acid sequence.

In another aspect of this invention, there is provided a method fordetecting a target nucleic acid sequences from a DNA or a mixture ofnucleic acids by a PTOCE (PTO Cleavage and Extension) assay, comprising:

-   -   (a) hybridizing the target nucleic acid sequences with a primer        pair comprising an upstream primer and a downstream primer and a        PTO (Probing and Tagging Oligonucleotide); wherein each of the        upstream primer and the downstream primer comprise a hybridizing        nucleotide sequence complementary to the target nucleic acid        sequence; the PTO comprises (i) a 3′-targeting portion        comprising a hybridizing nucleotide sequence complementary to        the target nucleic acid sequence and (ii) a 5′-tagging portion        comprising a nucleotide sequence non-complementary to the target        nucleic acid sequence; wherein the 3′-targeting portion is        hybridized with the target nucleic acid sequence and the        5′-tagging portion is not hybridized with the target nucleic        acid sequence; the PTO is located between the upstream primer        and the downstream primer; wherein the PTO is blocked at its        3′-end to prohibit its extension;    -   (b) contacting the resultant of the step (a) to a        template-dependent nucleic acid polymerase having a 5′ nuclease        activity under conditions for extension of the primers and for        cleavage of the PTO; wherein when the PTO is hybridized with the        target nucleic acid sequences, the upstream primer is extended        and the extended strand induces cleavage of the PTO by the        template-dependent nucleic acid polymerase having the 5′        nuclease activity such that the cleavage releases a fragment        comprising the 5′-tagging portion or a part of the 5′-tagging        portion of the PTO;    -   (c) hybridizing the fragment released from the PTO with a CTO        (Capturing and Templating Oligonucleotide); wherein the CTO        comprises in a 3′ to 5′ direction (i) a capturing portion        comprising a nucleotide sequence complementary to the 5′-tagging        portion or a part of the 5′-tagging portion of the PTO and (ii)        a templating portion comprising a nucleotide sequence        non-complementary to the 5′-tagging portion and the 3′-targeting        portion; wherein the fragment released from the PTO is        hybridized with the capturing portions of the CTO;    -   (d) performing an extension reaction using the resultant of the        step (c) and the template-dependent nucleic acid polymerase;        wherein the fragment hybridized with the capturing portion of        the CTO is extended and an extended duplex is formed; wherein        the extended duplex has a T_(m) value adjustable by (i) a        sequence and/or length of the fragment, (ii) a sequence and/or        length of the CTO or (iii) the sequence and/or length of the        fragment and the sequence and/or length of the CTO;    -   (e) melting the extended duplex over a range of temperatures to        give a target signal indicative of the presence of the extended        duplex; wherein the target signal is provided by (i) at least        one label linked to the fragment and/or the CTO, (ii) a label        incorporated into the extended duplex during the extension        reaction, (iii) a label incorporated into the extended duplex        during the extension reaction and a label linked to the fragment        and/or the CTO, or (iv) intercalating label; and    -   (f) detecting the extended duplex by measuring the target        signal; whereby the presence of the extended duplex indicates        the presence of the target nucleic acid sequence.

Since the preferable embodiment of the present invention follows thesteps of the present method described above, the common descriptionsbetween them are omitted in order to avoid undue redundancy leading tothe complexity of this specification.

According to a preferred embodiment, the method further compriserepeating the steps (a)-(b), (a)-(d) or (a)-(f) with denaturationbetween repeating cycles. The reaction repetition is accompanied withamplification of the target nucleic acid sequence. Preferably, theamplification is performed in accordance with PCR (polymerase chainreaction) which is disclosed in U.S. Pat. Nos. 4,683,195, 4,683,202, and4,800,159.

According to a preferred embodiment, the method is performed to detectat least two types of target nucleic acid sequences.

According to a preferred embodiment, the at least two type of the targetsignals corresponding to the at least two types of the target nucleicacid sequences are provided from the same type of labels; wherein theextended duplexes corresponding to the at least two types of the targetnucleic acid sequences have different T_(m) values from each other.

III. Target Detection Process by PTOCE Comprising Detection at aPre-Determined Temperature

The present invention can be modified to utilize a target signalgenerated in association with the formation of the extended duplex.

In still another aspect of this invention, there is provided a methodfor detecting a target nucleic acid sequence from a DNA or a mixture ofnucleic acids by a PTOCE (PTO Cleavage and Extension) assay, comprising:

-   -   (a) hybridizing the target nucleic acid sequence with an        upstream oligonucleotide and a PTO (Probing and Tagging        Oligonucleotide); wherein the upstream oligonucleotide comprises        a hybridizing nucleotide sequence complementary to the target        nucleic acid sequence; the PTO comprises (i) a 3′-targeting        portion comprising a hybridizing nucleotide sequence        complementary to the target nucleic acid sequence and (ii) a        5′-tagging portion comprising a nucleotide sequence        non-complementary to the target nucleic acid sequence; wherein        the 3′-targeting portion is hybridized with the target nucleic        acid sequence and the 5′-tagging portion is not hybridized with        the target nucleic acid sequence; the upstream oligonucleotide        is located upstream of the PTO;    -   (b) contacting the resultant of the step (a) to an enzyme having        a 5′ nuclease activity under conditions for cleavage of the PTO;        wherein the upstream oligonucleotide or its extended strand        induces cleavage of the PTO by the enzyme having the 5′ nuclease        activity such that the cleavage releases a fragment comprising        the 5′-tagging portion or a part of the 5′-tagging portion of        the PTO;    -   (c) hybridizing the fragment released from the PTO with a (ID        (Capturing and Templating Oligonucleotide); wherein the CTO        comprises in a 3′ to 5 direction (i) a capturing portion        comprising a nucleotide sequence complementary to the 5′-tagging        portion or a part of the 5′-tagging portion of the PTO and (ii)        a templating portion comprising a nucleotide sequence        non-complementary to the 5′-tagging portion and the 3′-targeting        portion of the PTO; wherein the fragment released from the PTO        is hybridized with the capturing portion of the CTO;    -   (d) performing an extension reaction using the resultant of the        step (c) and a template-dependent nucleic acid polymerase;        wherein the fragment hybridized with the capturing portion of        the CTO is extended to form an extended duplex; wherein the        extended duplex has a T_(m) value adjustable by (i) a sequence        and/or length of the fragment, (ii) a sequence and/or length of        the CTO or (iii) the sequence and/or length of the fragment and        the sequence and/or length of the CTO; wherein the extended        duplex provides a target signal by (i) at least one label linked        to the fragment and/or CTO, (ii) a label incorporated into the        extended duplex during the extension reaction, (iii) at least        one label linked to the fragment and/or CTO and a label        incorporated into the extended duplex during the extension        reaction or (iv) intercalating label; and    -   (e) detecting the extended duplex by measuring the target signal        at a predetermined temperature that the extended duplex        maintains its double-stranded form, whereby the presence of the        extended duplex indicates the presence of the target nucleic        acid sequence.

Since the preferable embodiment of the present invention follows thesteps of the present method above-described except for the melting step,the common descriptions between them are omitted in order to avoid undueredundancy leading to the complexity of this specification.

The present invention using a melting analysis described hereinaboverequires detection of signals from labels at not less than two differenttemperatures because the target signal is given by measuring signalchange provided in melting of the extended duplex.

Unlikely, in this aspect of this invention, the extended duplex per segives signal capable of discriminating formation from no-formation ofthe extended duplex and the signal is detected at a predeterminedtemperature that the extended duplex maintains its double-stranded form,whereby the presence of a target nucleic acid sequence is determined.

The present invention is to measure a target signal in association withthe formation of the extended duplex, for detection of the presence ofthe target nucleic acid sequence.

In the present invention, the extended duplex has a label such that theextended duplex provides a target signal.

Preferably, the target signal includes a signal (signal generation orsignal extinguishment) from the label on the extended duplex at apre-determined temperature.

The labeling in the present invention may be executed in the same manneras that for the method using a melting analysis described above. FIGS.2-13 may illustrate this aspect of the present invention with a littlemodification for detection at a pre-determined temperature.

The working principle underlying a target signal from the extendedduplex is as follows: (i) the extension of the fragment induces changeof a signal from a label to give the target signal; or

(ii) the hybridization of the fragment and the CTO induces change of asignal from a label to give the target signal and the extended duplexmaintains the target signal.

The exemplified embodiment of the working principle (i) may be describedwith referring to FIG. 9. Where immobilized CTOs are used, the presentinvention detects a plurality of target nucleic acid sequences in muchmore effective manner. The templating portion of the immobilized CTO hasa reporter molecule and a quencher molecule. The reporter molecule andthe quencher molecule are conformationally adjacent to each other toallow the quencher molecule to quench a signal from the reportermolecule. When the fragment is hybridized with the capturing portion ofthe CTO, the quencher molecule quenches the signal from the reportermolecule. By the formation of the extended duplex, the reporter moleculeand the quencher molecule are conformationally separated to allow thequencher molecule to unquench the signal from the reporter molecule. Thetarget signal is given in the extension step (C and D in FIG. 9).

In FIG. 9, the hybrid between the uncleaned PTO and CTO does not form anextended duplex. Therefore, the quencher molecule is allowed to stillquench a signal from the reporter molecule. The hybrid does not providenon-target signal.

The exemplified embodiment for the working principle (ii) may bedescribed with referring to FIG. 6. The figure illustrates the presentaspect as well as the method using melting analysis. The 5′-taggingportion of the PTO has a reporter molecule and a quencher molecule. Thereporter molecule and the quencher molecule are conformationallyadjacent to each other to allow the quencher molecule to quench a signalfrom the reporter molecule. The PTO hybridized with the target nucleicacid sequence is digested to release the fragment comprising the5′-tagging portion with the reporter molecule and the quencher molecule,and the fragment is hybridized with the capturing portion of the CTO. Bythe hybridization, the reporter molecule and the quencher molecule areconformationally separated to allow the quencher molecule to unquenchthe signal from the reporter molecule. The target signal is given in thefragment hybridization step and the extended duplex maintains the targetsignal (C and D in FIG. 6).

In FIG. 6, the hybrid between the uncleaved PTO and the CTO provides nontarget signal (C and D in FIG. 6) and it is necessary to dissociate thehybrid to remove the non-target signal. Therefore, the temperature formeasuring the target signal is determined to dissociate the hybrid.According to a preferred embodiment, the temperature is furtherdetermined in consideration of hybrid's T_(m) value.

According to a preferred embodiment, the extended duplex may be detectedat temperatures that the hybrid is partially dissociated.

The predetermined temperature is higher than the hybrid's T_(m) valueminus 10° C., preferably, higher than the hybrid's T_(m) value minus 5°C., more preferably, higher than the hybrid's T_(m) value and still morepreferably, higher than the hybrid's T_(m) value plus 5° C.

According to a preferred embodiment, the target signal provided by theextended duplex is given during the extension of the step (d); wherein ahybrid between an uncleaved PTO and the CTO does not provides anon-target signal, as represented in FIGS. 2-4 and 9-11.

According to a preferred embodiment, the target signal provided by theextended duplex is given by the hybridization of the fragment and theCTO in the step (c) and the formation of the extended duplex maintainsthe target signal in the step (d); wherein a hybrid between an uncleavedPTO and the CTO provides a non-target signal; wherein the predeterminedtemperature is higher than the hybrid's T_(m) value, as represented inFIGS. 5-8 and 12-13.

When the hybrid between the cleaved PTO and CTO provides non-targetsignal (Panel D in FIG. 6), it is necessary to dissociate the hybrid toremove the non target signal. Therefore, the temperature for measuringtarget signal is determined to dissociate the hybrid.

The labeling systems useful in this invention will be described asfollows:

(i) Label Linked to the Fragment and/or the CTO

(i-4) Interactive Dual Label

In an embodiment of an interactive dual label system, the CTO has aninteractive dual label comprising a reporter molecule and a quenchermolecule; wherein the extension of the fragment in the step (d) induceschange of a signal from the interactive dual label to give the targetsignal. The first embodiment of the interactive dual label system isillustrated in FIG. 2. The target signal is given withextension-synchronized signal generation.

According to a preferred embodiment, the reporter molecule and thequencher molecule may be located at the templating portion of the CTO.

According to a preferred embodiment, one of the reporter molecule andthe quencher molecule on the CTO is located at its 5′-end or at 1-5nucleotides apart from its 5′-end and the other is located to quench andquench the signal from the reporter molecule depending on conformationof CTO

In an embodiment of an interactive dual label system, the CTO has aninteractive dual label comprising a reporter molecule and a quenchermolecule; wherein the hybridization of the fragment and the CTO in thestep (c) induces change of a signal from the interactive dual label togive the target signal and the extended duplex maintains the targetsignal.

According to the preferred embodiment, the reporter molecule and thequencher molecule may be located at the capturing portion of the CTO.

According to the preferred embodiment, one the reporter molecule and thequencher molecule on the CTO is located at its 3′-end or at 1-5nucleotides apart from its 3′-end and the other is located to quench andunquench the signal from the reporter molecule depending on conformationof CTO.

In this embodiment, the hybrid between the uncleaved PTO and the CTOprovides non-target signal; wherein the temperature for measuring thetarget signal is determined with consideration of the Tm value of thehybrid.

In an embodiment of an interactive dual label system, the fragment hasan interactive dual label comprising a reporter molecule and a quenchermolecule; wherein the hybridization of the fragment and the CTO in thestep (e) induces change of a signal from the interactive dual label togive the target signal and the extended duplex maintains the targetsignal. The first embodiment of the interactive dual label system isillustrated in FIG. 6.

According to the preferred embodiment, one of the reporter molecule andthe is quencher molecule on the fragment is located at its 5′-end or at1-5 nucleotides apart from the 5′-end of the fragment and the other islocated to quench the signal from the reporter molecule depending onconformation of the fragment.

In this embodiment, the hybrid between the uncleaved PTO and the CTOprovides non-target signal; wherein the temperature for measuring thetarget signal is determined with consideration of the Tm value of thehybrid.

In an embodiment of the interactive label system, wherein the fragmenthas one of an interactive dual label comprising a reporter molecule anda quencher molecule and the CTO has the other of the interactive duallabel; wherein the hybridization of the fragment and the CTO in the step(c) induces change of a signal from the interactive dual label to givethe target signal and the extended duplex maintains the target signal.The embodiment of the interactive dual label system is illustrated inFIG. 8.

The reporter molecule and the quencher molecule may be located at anysite of the PTO fragment and the CTO, so long as the signal from thereporter molecule is quenched by the quencher molecule.

According to the embodiment, the reporter molecule or the quenchermolecule on the PTO fragment is located, preferably, at its 5′-end.

According to the embodiment, the reporter molecule or the quenchermolecule on the CTO is located, preferably, at its 5′-end.

In this embodiment, the hybrid between the uncleaved PTO and the CTOprovides non-target signal; wherein the temperature for measuring thetarget signal is determined with consideration of the Tm value of thehybrid.

(i-2) Single Label

In an embodiment of a single label system, the CTO has a single labeland the extension of the fragment in the step (d) induces change of asignal from the single label to give the target signal. The embodimentof the single label system is illustrated in FIG. 3. The target signalis given with extension-synchronized signal generation.

According to the embodiment, the templating portion of the CTO islabeled with the single label.

In an embodiment of a single label system, the CTO has a single labeland the hybridization of the fragment and the CTO in the step (c)induces change of a signal from the interactive dual label to give thetarget signal and the extended duplex maintains the target signal.

According to the embodiment, the capturing portion of the CTO is labeledwith the single label.

In this embodiment, the hybrid between the uncleaved PTO and the CTOprovides non-target signal; wherein the temperature for measuring thetarget signal is determined with consideration of the Tm value of thehybrid.

In an embodiment of a single label system, the fragment has a singlelabel and the hybridization of the fragment and the CTO in the step (c)induces change of a signal from the interactive dual label to give thetarget signal and the extended duplex maintains the target signal. Theembodiment of the single label system is illustrated in FIG. 12.

In this embodiment, the hybrid between the uncleaved PTO and the CTOprovides non-target signal; wherein the temperature for measuring thetarget signal is determined with consideration of the Tm value of thehybrid.

The single label used herein has to be capable of providing a differentsignal depending on its presence on double strand or single strand. Thesingle label includes a fluorescent label, a luminescent label, achemiluminescent label, an electrochemical label and a metal label.Preferably, the single label includes a fluorescent label. The types andpreferable binding sites of single fluorescent labels used in thisinvention are disclosed U.S. Pat. Nos. 7,537,886 and 7,348,141, theteachings of which are incorporated herein by reference in their entity.Preferably, the single fluorescent label includes JOE, FAM, TAMRA, ROXand fluorescein-based label. The labeled nucleotide residue ispreferably positioned internal nucleotide residue within theoligonucleotide rather than at the 5′-end or the 3′-end.

The single fluorescent label useful in the present invention may bedescribed with reference to descriptions for reporter and quenchermolecules as indicated above.

In particular, where the present invention on a solid phase is performedusing a single label, it can utilize a general fluorescent label anddoes not require a specific fluorescent label capable of providing afluorescent signal with different intensities depending on its presenceon double strand or single strand.

When the CTO immobilized onto a solid substrate is used, chemical labels(e.g. biotin) or enzymatic labels (e.g. alkaline phosphatase,peroxidase, β-galactosidase and β-glucosidase) may be used.

In a preferred embodiment, the labels linked to the fragment and/or theCTO are positioned to the extent that when a hybrid between an uncleavedPTO and the CTO is formed, the hybrid does not give a non-target signalin the step (d), as represented in FIGS. 2-3 and 9.

Alternatively, the labels may be positioned to the extent that when ahybrid between an uncleaved PTO and the CTO is formed, the hybrid givesa non-target signal in the step (d); wherein the T_(m) value of theextended duplex is higher than that of the hybrid between the uncleavedPTO and the CTO as represented in FIGS. 6-8 and 12.

(ii) Label Incorporated into the Extended Duplex

In particular, where the present invention is carried out in a solidphase using an immobilized CTO, this label system becomes more useful toprovide the target signal as illustrated in FIGS. 10 and 11.

According to a preferred embodiment, the target signal is provided by asingle label incorporated into the extended duplex during the extensionreaction; wherein the incorporated single label is linked to anucleotide incorporated during the extension reaction; wherein theextension of the fragment in the step (d) induces change of a signalfrom the single label to give the target signal in the step (d).

According to a preferred embodiment, the nucleotide incorporated duringthe extension reaction has a first non-natural base and the CTO has anucleotide having a second non-natural base with a specific bindingaffinity to the first non-natural base, as illustrated in FIG. 11. Thenucleotide having the second non-natural base is preferably located atany site on the templating portion of the CTO.

Where the label incorporated into the extended duplex during theextension reaction is employed, the label is not incorporated into thehybrid between the uncleaved PTO and the CTO because the hybrid is notextended. Therefore, the hybrid does not provide a non-target signal.

(iii) Label Incorporated into the Extended Duplex and Label Linked tothe Fragment or the CTO

The present invention may employ a labeling system using cooperation ofa label incorporated into the extended duplex during the extensionreaction and a label linked to the fragment and/or the CTO, asillustrated in FIGS. 4 and 5.

According to a preferred embodiment, the target signal is provided by alabel incorporated into the extended duplex during the extensionreaction and a label linked to the fragment and/or the CTO; wherein thelabel incorporated is linked to a nucleotide incorporated during theextension reaction; wherein the two labels are an interactive dual labelof a reporter molecule and a quencher molecule; wherein the extension ofthe fragment in the step (d) induces change of a signal from theinteractive dual label to give the target signal.

More preferably, the nucleotide incorporated during the extensionreaction has a first non-natural base and the CTO has a nucleotidehaving a second non-natural base with a specific binding affinity to thefirst non-natural.

Preferably, the target signal given in the step (e) is a signal from theinteractive dual label in the step (d).

Where the label incorporated into the extended duplex during theextension reaction is employed, the label is not incorporated into thehybrid between the uncleaved PTO and the CTO because the hybrid is notextended. Therefore, the hybrid does not provide a non-target signal.

(iv) Intercalating Label

The present invention may employ an intercalating label for providingthe target signal indicative of the presence of the extended duplex. Theintercalating label is more useful on a solid phase reaction usingimmobilized CTOs because double-stranded nucleic acid molecules presentin samples can generate signals.

The exemplified embodiment is described with reference to FIG. 13. ThePTO hybridized with the target nucleic acid sequence is digested torelease the fragment. The fragment is hybridized with the CTO. Theextension is carried out in the presence of an intercalating dye (e.g.,SYBR™ Green) and forms the extended duplex with intercalating dyes.

In FIG. 13, the hybrid between the uncleaved PTO and the CTO providesnon-target signal (C and D in FIG. 13) and it is necessary to dissociatethe hybrid to remove the non-target signal. Therefore, the temperaturefor measuring the target signal is determined with consideration of theTm value of the hybrid.

Preferably, the target signal given in the step (e) is a signal from theintercalated dye.

According to a preferred embodiment, the PTO and/or CTO is blocked atits 3′-end to prohibit its extension.

According to a preferred embodiment, the upstream oligonucleotide is anupstream primer or an upstream probe.

According to a preferred embodiment, the upstream oligonucleotide islocated adjacently to the PTO to the extent that the upstreamoligonucleotide induces cleavage of the PTO by the enzyme having the 5′nuclease activity.

According to a preferred embodiment, the upstream primer induces throughits extended strand the cleavage of the PTO by the enzyme having the 5′nuclease activity.

According to a preferred embodiment, the method further comprisesrepeating the steps (a)-(b), (a)-(d) or (a)-(e) with denaturationbetween repeating cycles.

According to a preferred embodiment, the steps (a)(b) and (c)-(e) areperformed in a reaction vessel or in separate reaction vessels.

According to a preferred embodiment, the method is performed to detectat least two types of target nucleic acid sequences; wherein theupstream oligonucleotide comprises at least two types ofoligonucleotides, the PTO comprises at least two types of the PTOs, andthe CTO comprises at least one type of the CTOs; wherein when at leasttwo types of the target nucleic acid sequences are present, the methodprovides at least two types of the target signals corresponding to theat least two types of the target nucleic acid sequences.

According to a preferred embodiment, the upstream oligonucleotide is anupstream primer and the step (b) uses a template-dependent nucleic acidpolymerase for the extension of the upstream primer.

According to a preferred embodiment, the CTO is immobilized through its5′-end or 3′-end onto a solid substrate and the target signal providedon the solid substrate is measured.

According to a preferred embodiment, the target signal is provided by asingle label linked to the fragment or by a sing label incorporated intothe extended duplex during the extension reaction.

According to a preferred embodiment, the method is performed in thepresence of a downstream primer.

The detection of the step (e) may be performed in a real-time manner, anend point manner, or a predetermined time interval manner. Where thepresent invention further comprises repeating the steps (a)-(b), (a)-(d)or (a)-(e), it is preferred that the signal detection is performed foreach cycle of the repetition at a predetermined temperature (i.e.real-time manner), at the end of the repetition at a predeterminedtemperature (i.e. end-point manner) or at each of predetermined timeintervals during the repetition at a predetermined temperature.Preferably, the detection may be performed for each cycle of therepetition in a real-time manner to improve the detection accuracy andquantification.

IV. Kits for Target Detection

In further aspect of this invention, there is provided a kit fordetecting a target nucleic acid sequence from a DNA or a mixture ofnucleic acids by a PTOCE (PTO Cleavage and Extension) assay, comprising:

-   -   (a) an upstream oligonucleotide comprising a hybridizing        nucleotide sequence complementary to the target nucleic acid        sequence;    -   (b) a PTO (Probing and Tagging Oligonucleotide) comprising (i) a        3′-targeting portion comprising a hybridizing nucleotide        sequence complementary to the target nucleic acid sequence        and (ii) a 5′-tagging portion comprising a nucleotide sequence        non-complementary to the target nucleic acid sequence, wherein        the 3′-targeting portion is hybridized with the target nucleic        acid sequence and the 5′-tagging portion is not hybridized with        the target nucleic acid sequence; the upstream oligonucleotide        is located upstream of the PTO; wherein the upstream        oligonucleotide or its extended strand induces cleavage of the        PTO by an enzyme having a 5′ nuclease activity such that the        cleavage releases a fragment comprising the 5′-tagging portion        or a part of the 5′-tagging portion of the PTO; and    -   (c) a CTO (Capturing and Templating Oligonucleotide) comprising        in a 3′ to 5′ direction (i) a capturing portion comprising a        nucleotide sequence complementary to the 5′-tagging portion or a        part of the 5′-tagging portion of the PTO and (ii) a templating        portion comprising a nucleotide sequence non-complementary to        the 5′-tagging portion and the 3′-targeting portion of the PTO;        wherein the fragment released from the PTO is hybridized with        the capturing portion of the CTO; and the fragment hybridized        with the capturing portion of the CTO is extended by a        template-dependent nucleic acid polymerase to form an extended        duplex.

Since the kit of this invention is constructed to perform the detectionmethod of the present invention described above, the common descriptionsbetween them are omitted in order to avoid undue redundancy leading tothe complexity of this specification.

According to a preferred embodiment, the kit further comprises an enzymehaving a 5′ nuclease activity.

According to a preferred embodiment, the kit further comprises atemplate-dependent nucleic acid polymerase.

According to a preferred embodiment, the PTO and/or the CTO has at leastone label.

According to a preferred embodiment, the kit further comprises a labelto be incorporated into the extended duplex during the extensionreaction.

According to a preferred embodiment, the kit further comprises a labelto be incorporated into the extended duplex during the extensionreaction and the PTO and/or the CTO has at least one label.

According to a preferred embodiment, the kit further comprises anintercalating label.

According to a preferred embodiment, the label is a single label orinteractive dual label.

According to a preferred embodiment, the kit is used for detection of atleast two types of nucleic acid sequences, the upstream oligonucleotidecomprises at least two types of oligonucleotides, the PTO comprises atleast two types of the PTO and the CTO comprises at least two types ofthe CTO.

According to a preferred embodiment, the CTO is immobilized through its5′-end or 3′-end onto a solid substrate.

According to a preferred embodiment, the kit further comprises adownstream primer.

All of the present kits described hereinabove may optionally include thereagents required for performing target amplification PCR reactions(e.g., PCR reactions) such as buffers, DNA polymerase cofactors, anddeoxyribonucleotide-5-triphosphates. Optionally, the kits may alsoinclude various polynucleotide molecules, reverse transcriptase, variousbuffers and reagents, and antibodies that inhibit DNA polymeraseactivity. The kits may also include reagents necessary for performingpositive and negative control reactions. Optimal amounts of reagents tobe used in a given reaction can be readily determined by the skilledartisan having the benefit of the current disclosure. The kits,typically, are adopted to contain the constituents afore-described inseparate packaging or compartments.

The features and advantages of this invention will be summarized asfollows:

(a) The present invention provides a target-dependent extended duplex inwhich PTO (Probing and Tagging Oligonucleotide) hybridized with a targetnucleic acid sequence is cleaved to release a fragment and the fragmentis hybridized with CTO (Capturing and Templating Oligonucleotide) toform an extended duplex. The extended duplex provides a signal (signalgeneration or extinguishment) or a signal change (signal increase ordecrease) indicating the presence of a target nucleic acid sequence.

(b) The presence of the extended duplex is determined by a variety ofmethods or processes such as melting curve analysis and detection at apre-determined temperature (e.g. a real-time manner and end-pointmanner).

(c) The present invention allows to simultaneously detect at least twotypes of target nucleic acid sequences by melting curve analysis evenusing a single type of a label (e.g. FAM). In contrast, the conventionalmultiplex real-time method performed in a liquid phase is seriouslysuffering from limitation associated with the number of detectablefluorescence labels. The present invention permits to successfullyovercome such shortcomings and widen the application of multiplexreal-time detection.

(d) The present invention can be performed using a multitude of labelingsystems. For example, the labels linked to any site of PTO and/or CTOcan be utilized for providing the target signal indicating the extendedduplex. Also, labels incorporated into the extended duplex during theextension reaction can be used in the present invention. In addition tothis, a combination of such labels can be used. The versatile labelingsystems applicable to the present invention allow us to choose a properlabeling system depending on experimental conditions or objectives.

(e) The present invention provides a target-dependent extended duplexwhich has a pre-determined T_(m) value adjustable by (i) a sequenceand/or length of the fragment, (ii) a sequence and/or length of the CTOor (iii) the sequence and/or length of the fragment and the sequenceand/or length of the CTO.

(f) Conventional melting curve analysis using an amplified productdepends on the sequence of the amplified product such that it isdifficult to obtain a desired Tm value of amplified product. Incontrast, the present invention depends on the sequence of an extendedduplex not the sequence of an amplified product, permitting to select adesired T_(m) value of extended duplex. Therefore, the present inventionis easily adoptable for the detection of multiple target sequences.

(g) Conventional melting curve analysis using a direct hybridizationbetween labeled probes and target nucleic acid sequences is very likelyto generate false positive signals due to non-specific hybridization ofprobes. In contrast, the present invention employs not only PTOhybridization but also enzymatic cleavage and extension, which overcomescompletely problems of false positive signals.

(h) Tm value of conventional melting curve analysis is affected by asequence variation on the target nucleic acid sequences. However, anextended duplex in the present invention provides a constant Tm valueregardless of a sequence variation on the target nucleic acid sequences,permitting to ensure excellent accuracy in melting curve analysis.

(i) it is noteworthy that the sequence of the 5′-tagging portion of PTOand the sequence of CTO can be selected with no consideration of targetnucleic acid sequences. This makes it possible to pre-design a pool ofsequences for the 5′-tagging portion of PTO and CTO. Although the3′-targeting portion of the PTO has to be prepared with consideringtarget nucleic acid sequences, the CTO can be prepared in a ready-madefashion with no consideration or knowledge of target nucleic acidsequences. Such features provide prominent advantages in multiple targetdetection, inter alia, on a microarray assay using CTOs immobilized ontoa solid substrate.

The present invention will now be described in further detail byexamples. It would be obvious to those skilled in the art that theseexamples are intended to be more concretely illustrative and the scopeof the present invention as set forth in the appended claims is notlimited to or by the examples.

EXAMPLES Example 1 Evaluation of Probing and Tagging OligonucleotideCleavage & Extension (PTOCE) Assay

A New assay, Probing and Tagging Oligonucleotide Cleavage & Extension(PTOCE) assay, was evaluated whether an extended duplex can provide atarget signal for the detection of a target nucleic acid sequence.

For this evaluation, PTOCE assay detecting the presence of an extendedduplex by melting analysis was performed (PTOCE assay comprising meltinganalysis). We used Taq DNA polymerase having a 5′ nuclease activity forthe extension of upstream primer, the cleavage of PTO and the extensionof PTO fragment.

The extended duplex formed during the assay was designed to have aninteractive dual label. The interactive dual label in the extendedduplex was provided by (i) CTO labeled with a reporter molecule and aquencher molecule (dual-labeled CTO) or (ii) PTO having a quenchermolecule and CTO having a reporter molecule (a quencher-labeled PTO anda reporter-labeled CTO). PTO and CTO are blocked with a carbon spacer attheir 3′-ends. The synthetic oligonucleotide for Neisseria gonorrhoeae(NG) gene was used as a target template.

1-1. PTOCE Assay Using a Dual-Labeled CTO

PTO has no label. CTO has a quencher molecule (BHQ-1) and a fluorescentreporter molecule (FAM) in its templating portion. The sequences ofsynthetic template, upstream primer, PTO and CTO used in this Exampleare:

NG-T (SEQ ID NO: 1) 5′-AAATATGCGAACACGCCAATGAGGGGCATGATGCTTTCTTTTTGTTCTTGCTCGGCAGAGCGAGTGATACCGATCCATTGAAAAA-3′ NG-R (SEQ ID NO: 2)5′-CAATGGATCGGTATCACTCGC-3′ NG-PTO-1 (SEQ ID NO: 3)5′-ACGACGGCTTGGCTGCCCCTCATTGGCGTGTTTCG[C3 spacer]-3′ NG-CTO-1(SEQ ID NO: 4) 5′-[BHQ-1]CCTCCTCCTCCTCCTCCTCC[T(FAM)]CCAGTAAAGCCAAGCCGTCGT[C3 Spacer]-3′ (Underlined letters indicate the 5′-taggingportion of PTO)

The reaction was conducted in the final volume of 20 μl containing 2pmole of synthetic template (SEQ ID NO: 1) for NG gene, 10 mole ofupstream primer (SEQ ID NO: 2), 5 pmole of PTO (SEQ ID NO: 3), 2 pmoleof CTO (SEQ ID NO: 4) and 10 μl of 2× Master Mix containing 2.5 mMMgCl₂, 200 μM of dNTPs and 1.6 units of H-Taq DNA polymerase (Solgent,Korea); the tube containing the reaction mixture was placed in thereal-time thermocycler (CFX96, Bio-Rad); the reaction mixture wasdenatured for 15 min at 95° C. and subjected to 30 cycles of 30 sec at95° C., 60 sec at 60° C. After the reaction, melting curve was obtainedby cooling the reaction mixture to 35° C., holding at for 35° C. for 30sec, and heating slowly at 35° C. to 90° C. The fluorescence wasmeasured continuously during the temperature rise to monitordissociation of double-stranded DNAs. Melting peak was derived from themelting curve data.

As shown FIG. 14, a peak at 76.5° C. corresponding to the expected Tmvalue of the extended duplex was detected in the presence of thetemplate. No peak was detected in the absence of the template. Since thehybrid of uncleaved PTO and CTO does not give any signal in thislabeling method, there was no peak corresponding to the hybrid ofuncleaved PTO and CTO. In case of no PTO or no CTO, any peak was notobserved.

1-2. PTOCE Assay Using a Quencher-Labeled PTO and a Reporter-Labeled CTO

PTO is labeled with a quencher molecule (BHQ-1) at its 5′-end. CTO islabeled with a fluorescent reporter molecule (FAM) at its 3′-end.

The sequences of synthetic template, upstream primer, PTO and CTO usedin this Example are:

NG-T (SEQ ID NO: 1) 5′-AAATATGCGAAACACGCCAATGAGGGGCATGATGCTTTCTTTTTGTTCTTGCTCGGCAGAGCGAGTGATACCGATCCATTGAAAAA-3′ NG-R (SEQ ID NO: 2)5′-CAATGGATCGGTATCACTCGC-3′ NG-PTO-2 (SEQ ID NO: 5)5′-[BHQ-1]ACGACGGCTTGGCTTTACTGCCCCTCATTGGCGTGTT TCG[C3 spacer]-3′NG-CTO-2 (SEQ ID NO: 6) 5′-CCTCCTCCTCCTCCTCCTCCTCCAGTAAAGCCAAGCCGTCGT[FAM]-3′ (Underlined letters indicate the 5′-tagging  portion of PTO)

The reaction was conducted in the final volume of 20 μl containing 2pmole of synthetic template (SEQ ID NO: 1) for NG gene, 10 pmole ofupstream primer (SEQ ID NO: 2), 5 pmole of PTO (SEQ ID NO: 5), 2 pmoleof CTO (SEQ ID NO: 6) and 10 μl of 2× Master Mix containing 2.5 mMMgCl₂, 200 μM of dNTPs and 1.6 units of H-Taq DNA polymerase (Solgent,Korea); the tube containing the reaction mixture was placed in thereal-time thermocycler (CFX96, Bio-Rad); the reaction mixture wasdenatured for 15 min at 95° C. and subjected to 30 cycles of 30 sec at95° C., 60 sec at 60° C., 30 sec at 72° C. After the reaction, meltingcurve was obtained by cooling the reaction mixture to 35° C., holding atfor 35° C. for 30 sec, and heating slowly at 35° C. to 90° C. Thefluorescence was measured continuously during the temperature rise tomonitor dissociation of double-stranded DNAs. Melting peak was derivedfrom the melting curve data.

As shown FIG. 15, a peak at 77.0° C. corresponding to the expected Tmvalue of the extended duplex was detected in the presence of thetemplate. Since the hybrid of uncleaved PTO and CTO does give anon-target signal in this labeling method, there was a peak at 64.0°C.˜64.5° C. corresponding to the expected Tm value of the hybrid ofuncleaved PTO and CTO. In case of no PTO or no CTO, any peak was notobserved.

These results indicate that a target-dependent extended duplex isproduced and the extended duplex provides the target signal indicatingthe presence of the target nucleic acid sequence.

Example 2 Adjustability of Tm Value of an Extended Duplex

We further examined whether the Tm value of an extended duplex isadjustable by the sequence of CTO in PTOCE assay.

For the examination, we used three types of CTOs having differentsequences at their templating portions. PTO has no label. The threetypes of CTOs have a quencher molecule (BHQ-1) and a fluorescentreporter molecule (FAM) in their templating portions. PTO and CTO areblocked with a carbon spacer at their 3′-ends.

PTOCE assay comprising melting analysis was performed with each of thethree types of CTOs.

The sequences of synthetic template, upstream primer, PTO and CTOs usedin this Example are:

NG-T (SEQ ID NO: 1) 5′-AAATATGCGAAACACGCCAATGAGGGGCATGATGCTTTCTTTTTGTTCTTGCTCGGCAGAGCGAGTGATACCGATCCATTGAAAAA-3′ NG-R (SEQ ID NO: 2)5′CAATGGATCGGTATCACTCGC-3′ NG-PTO-3 (SEQ ID NO: 7)5′-ACGACGGCTTGGCCCCTCATTGGCGTGTTTCG[C3 spacer]-3′ NG-CTO-1(SEQ ID NO: 4) 5′-[BHQ-1]CCTCCTCCTCCTCCTCCTCC[T(FAM)]CCAGTAAAGCCAAGCCGTCGT[C3 Spacer]-3 NG-CTO-3 (SEQ ID NO: 8)5′-[BHQ-1]TTTTTTTTTTCCTCCTCCAG[T(FAM)]AAAGCCAAG CCGTCGT[C3 Spacer]-3′NG-CTO-4 (SEQ ID NO: 9) 5′-[BHQ-1]TTTTTTTTTTTTTTTTTTAG[T(FAM)]AAAGCCAAGCCGTCGT[C3 Spacer]-3′ (Underlined letters indicate the 5′-taggingportion of PTO)

The reaction was conducted in the final volume of 20 μl containing 2pmole of synthetic template (SEQ ID NO: 1) for NG gene, 10 pmole ofupstream primer (SEQ ID NO: 2), 5 mole of PTO (SEQ ID NO: 7), 2 pmole ofCTO (SEQ ID NOs: 4, 8, or 9), and 10 μl of 2× Master Mix containing 2.5mM MgCl₂, 200 μM of dNTPs and 1.6 units of H-Taq DNA polymerase(Solgent, Korea); the tube containing the reaction mixture was placed inthe real-time thermocycler (CFX96, Bio-Rad); the reaction mixture wasdenatured for 15 min at 95□ and subjected to 30 cycles of 30 sec at 95□,60 sec at 60□. After the reaction, melting curve was obtained by coolingthe reaction mixture to 35° C., holding at for 35° C. for 30 sec, andheating slowly at 35° C. to 90° C. The fluorescence was measuredcontinuously during the temperature rise to monitor dissociation ofdouble-stranded DNAs. Melting peak was derived from the melting curvedata.

As shown in FIG. 16, a peak was detected at 76.0° C., 69.0° C. or 64.5°C. in the presence of the template. Each peak corresponds to theexpected Tm of the extended duplex generated from the examined CTO. Nopeak was detected in the absence of the template.

These results indicate that the Tm value of the extended duplex isadjustable by the sequence of CTO.

Example 3 Detection of a Target Nucleic Acid Sequence Using PTOCE AssayComprising Real-Time Detection or Melting Analysis

We further examined whether the PTOCE assay can detect a target nucleicacid sequence in real-time PCR manner (i) or post-PCR melting analysismanner (ii): (i) Cleavage of PTO and extension of PTO fragment wereaccompanied with the amplification of a target nucleic acid by PCRprocess and the presence of the extended duplex was detected at apre-determined temperature, in each cycle (PTOCE assay comprisingreal-time detection at a pre-determined temperature) or; (ii) Cleavageof PTO and extension of PTO fragment were accompanied with theamplification of a target nucleic acid by PCR process and the presenceof the extended duplex was detected by post-PCR melting analysis (PTOCEassay comprising melting analysis).

Upstream primer is involved in the PTO cleavage by an enzyme having a 5′nuclease activity and also involved in amplification of the target acidsequence with downstream primer by PCR process. Taq DNA polymerasehaving a 5′ nuclease activity was used for the extension of upstreamprimer and downstream primer, the cleavage of PTO and the extension ofPTO fragment.

The extended duplex was designed to have an interactive dual label. Theinteractive dual label in the extended duplex was provided by (i) CTOlabeled with a reporter molecule and a quencher molecule, (ii) aquencher-iso-dGTP incorporated during extension reaction and CTO havinga reporter molecule and an iso-dC residue or (iii) PTO having a quenchermolecule and CTO having a reporter molecule. PTO and CTO are blockedwith a carbon spacer at their 3′-ends.

Genomic DNA of Neisseria gonorrhoeae (NG) was used as a target nucleicacid.

3-1. PTOCE Assay Using a Dual-Labeled CTO

PTO has no label and CTO is labeled with a quencher molecule (BHQ-1) anda fluorescent reporter molecule (FAM) in its templating portion.

The sequences of upstream primer, downstream primer, PTO and CTO used inthis Example are:

NG-F (SEQ ID NO: 10) 5′-TACGCCTGCTACTTTCACGCT-3′ NG-R (SEQ ID NO: 2)5′-CAATGGATCGGTATCACTCGC-3′ NG-PTO-3 (SEQ ID NO: 7)5′-ACGACGGCTTGGCCCCTCATTGGCGTGTTTCG[C3 spacer]-3′ NG-CTO-1(SEQ ID NO: 4) 5′-[BHQ-1]CCTCCTCCTCCTCCTCCTCC[T(FAM)]CCAGTAAAGCCAAGCCGTCG[C3 Spacer]-3′ (Underlined letters indicate the 5′-taggingportion of PTO)3-1-1. PTOCE Assay Comprising Real-Time Detection at a Pre-DeterminedTemperature

The reaction was conducted in the final volume of 20 μl containing 100pg of genomic DNA of NG, 10 pmole of downstream primer (SEQ ID NO: 10),10 pmole of upstream primer (SEQ ID NO: 2), 5 pmole of PTO (SEQ ID NO:7), 2 pmole of CTO (SEQ ID NO: 4), and 10 μl of 2× Master Mix containing2.5 mM MgCl₂, 200 μM of dNTPs and 1.6 units of H-Taq DNA polymerase(Solgent, Korea); the tube containing the reaction mixture was placed inthe real-time thermocycler (CFX96, Bio-Rad); the reaction mixture wasdenatured for 15 min at 95° C. and subjected to 60 cycles of 30 sec at95° C., 60 sec at 60° C., 30 sec at 72° C. Detection of the signal wasperformed at 60° C. of each cycle. The detection temperature wasdetermined to the extent that the extended duplex maintains adouble-stranded form.

As shown in FIG. 17A, the target signal (Ct 31.36) was detected in thepresence of the template. No signal was detected in the absence of thetemplate.

3-1-2. PTOCE Assay Comprising Melting Analysis

After the reaction in Example 3-1-1, melting curve was obtained bycooling the reaction mixture to 35° C., holding at for 35° C. for 30sec, and heating slowly at 35° C. to 90° C. The fluorescence wasmeasured continuously during the temperature rise to monitordissociation of double-stranded DNAs. Melting peak was derived from themelting curve data.

As shown FIG. 17B, a peak at 76.0° C. corresponding to the expected Tmvalue of the extended duplex was detected in the presence of thetemplate. No peak was detected in the absence of the template. Since thehybrid of uncleaved PTO and CTO does not give any signal in thislabeling method, there was no peak corresponding to the hybrid ofuncleaved PTO and CTO.

3-2. PTOCE Assay Using a Quencher-Iso-dGTP and a Reporter-Labeled CTOHaving an Iso-dC Residue

PTO has no label. CTO has a reporter molecule (FAM) and an iso-dCresidue at its 5′-end. During extension reaction of PTO fragment, aniso-dGTP labeled with a quencher molecule (dabcyl) is incorporated atthe position complementary to the iso-dC residue.

The sequences of upstream primer, downstream primer, PTO and CTO used inthis Example are:

NG-F (SEQ ID NO: 10) 5′-TACGCCTGCTACTTTCACGCT-3′ NG-R (SEQ ID NO: 2)5′-CAATGGATCGGTATCACTCGC-3′ NG-PTO-1 (SEQ ID NO: 3)5′-ACGACGGCTTGGCTGCVCCCTCATTGGCGTGTTTCG[C3 spacer]-3′ NG-CTO-5(SEQ ID NO: 11) 5′-[FAM][Iso-dC]CTCCTCCAGTAAAGCCAAGCCGTCGT[C3 spacer]-3′(Underlined letters indicate the 5′-tagging portion of PTO)3-2-1. PTOCE Assay Comprising Real-Time Detection at a Pre-DeterminedTemperature

The reaction was conducted in the final volume of 20 μl containing 100pg of genomic DNA of NG, 10 pmole of downstream primer (SEQ ID NO: 10),10 pmole of upstream primer (SEQ ID NO: 2), 5 pmole of PTO (SEQ ID NO:3), 2 pmole of CTO (SEQ ID NO: 11), and 10 μl of 2× Plexor® Master Mix(Cat. No. A4100, Promega, USA); the tube containing the reaction mixturewas placed in the real-time thermocycler (CFX96, Bio-Rad); the reactionmixture was denatured for 15 min at 95□ and subjected to 60 cycles of 30sec at 95□, 60 sec at 60□, 30 sec at 72□ and 5 cycles of 30 sec at 72□,30 sec at 55□. Detection of the signal was performed at 60□ of eachcycle. The detection temperature was determined to the extent that theextended duplex maintains a double-stranded form.

DNA polymerase having 5′ nuclease in the Plexor® Master Mix was used forthe extension of upstream primer and downstream primer, the cleavage ofPTO and the extension of PTO fragment.

As shown in FIG. 18A, the target signal (Ct 33.03) was detected in thepresence of the template. No signal was detected in the absence of thetemplate.

3-2-2. PTOCE Assay Comprising Melting Analysis

After the reaction in Example 3-2-1, melting curve was obtained bycooling the reaction mixture to 35° C., holding at for 35° C. for 30sec, and heating slowly at 35° C. to 90° C. The fluorescence wasmeasured continuously during the temperature rise to monitordissociation of double-stranded DNAs. Melting peak was derived from themelting curve data.

As shown FIG. 18B, a peak at 70.0° C. corresponding to the expected Tmvalue of the extended duplex was detected in the presence of thetemplate. No peak was detected in the absence of the template. Since thehybrid of uncleaved PTO and CTO does not give any signal in thislabeling method, there was no peak corresponding to the hybrid ofuncleaved PTO and CTO.

3-3. PTOCE Assay Using a Quencher-Labeled PTO and a Reporter-Labeled CTO

PTO is labeled with a quencher molecule (BHQ-1) at its 5′-end. CTO islabeled with a fluorescent reporter molecule (FAM) at its 3′-end.

The sequences of upstream primer, downstream primer, PTO and CTO used inthis Example are:

NG-F (SEQ ID NO: 10) 5′-TACGCCTGCTACTTTCACGCT-3′ NG-R (SEQ ID NO: 2)5′-CAATGGATCGGTATCACTCGC-3′ NG-PTO-4 (SEQ ID NO: 12)5′-[BHQ-1]ACGACGGCTTGCCCCTCATTGGCGTGTTTCG[C3 spacer]-3′ NG-CTO-2(SEQ ID NO: 6) 5′-CCTCCTCCTCCTCCTCCTCCTCCAGTAAAGCCAAGCCGTCGT [FAM]-3′(Underlined letters indicate the 5′-tagging portion of PTO)3-3-1. PTOCE Assay Comprising Real-Time Detection at a Pre-DeterminedTemperature

The reaction was conducted in the final volume of 20 μl containing 100pg of NG genomic DNA, 10 pmole of downstream primer (SEQ ID NO: 10), 10pmole of upstream primer (SEQ ID NO: 2), 5 pmole of PTO (SEQ ID NO: 12),2 pmole of CTO (SEQ ID NO: 6) and 10 μl of 2× Master Mix containing 2.5mM MgCl₂, 200 μM of dNTPs and 1.6 units of H-Taq DNA polymerase(Solgent, Korea); the tube containing the reaction mixture was placed inthe real-time thermocycler (CFX96, Bio-Rad); the reaction mixture wasdenatured for 15 min at 95° C. and subjected to 60 cycles of 30 sec at95° C., 60 sec at 60° C. 30 see at 72° C. Detection of the signal wasperformed at 60° C. of each cycle. The detection temperature wasdetermined to the extent that the extended duplex maintains adouble-stranded form and the temperature is higher than the T_(m) valueof a hybrid between uncleaved PTO and CTO.

As shown in FIG. 19A, the target signal (Ct 29.79) was detected in thepresence of the template. No signal was detected in the absence of thetemplate.

3-3-2, PTOCE Assay Comprising Melting Analysis

After the reaction in Example 3-3-1, melting curve was obtained bycooling the reaction mixture to 35° C., holding at for 35° C. for 30sec, and heating slowly at 35° C. to 90° C. The fluorescence wasmeasured continuously during the temperature rise to monitordissociation of double-stranded DNAs. Melting peak was derived from themelting curve data.

As shown FIG. 19B, a peak at 76.5° C. corresponding to the expected Tmvalue of the extended duplex was detected in the presence of thetemplate. Since the hybrid of uncleaved PTO and CTO does give anon-target signal in this labeling method, the peak corresponding to theTm value of the hybrid of uncleaved PTO and CTO was detected at 48.0° C.in the absence of the template.

These results indicate that a target nucleic acid sequence can bedetected by PTOCE assay comprising real-time detection or meltinganalysis.

Example 4 Detection of Multiple Target Nucleic Acid Sequences by PTOCEAssay Comprising Melting Analysis

We also examined whether the PTOCE assay comprising melting analysis candetect multiple target nucleic acid sequences using the same type of areporter molecule.

Cleavage of PTOs and extension of PTO fragments were accompanied withthe amplification of target nucleic acid sequences by PCR process andthe presence of the extended duplexes was detected by post-PCR meltinganalysis (PTOCE assay comprising melting analysis).

The extended duplexes formed during the assay were designed to have aninteractive dual label. The interactive dual label in extended duplexwas provided by CTO labeled with a reporter molecule and a quenchermolecule in its templating portion. The CTOs have the same type of afluorescent reporter molecule (PAM) but have different sequences togenerate the different Tm values of the extended duplexes. PTO and CTOare blocked with a carbon spacer at their 3′-ends.

Genomic DNAs of Neisseria gonorrhoeae (NG) and Staphylococcus aureus(SA) were used as target nucleic acids.

The sequences of upstream primer, downstream primer, PTOs and CTOs usedin this Example are:

NG-F (SEQ ID NO: 10) 5′-TACGCCTGCTACTTTCACGCT-3′ NG-R (SEQ ID NO: 2)5′-CAATGGATCGGTATCACTCGC-3′ NG-PTO-3 (SEQ ID NO: 7)5′-ACGACGGCTTGGCCCCTCATTGGCGTGTTTCG[C3 spacer]-3′ NG-CTO-1(SEQ ID NO: 4) 5′-[BHQ-1]CCTCCTCCTCCTCCTCCTCC[T(FAM)]CCAGTAAAGCCAAGCCGTCGT[C3 Spacer]-3′ SA-F (SEQ ID NO: 13)5′-TGTTAGAATTTGAACAAGGATTTAATC-3′ SA-R (SEQ ID NO: 14)5′-GATAAGTTTAAAGCTTGACCGTCTG-3′ SA-PTO-1 (SEQ ID NO: 15)5′-AATCCGACCACGCATTCCGTGGTCAATCATTCGGTTTACG[C3 spacer]-3′ SA-CTO-1(SEQ ID NO: 16) 5′-[BHQ-1]TTTTTTTTTTTTTTTTTGCA[T(FAM)]AGCGTGGTCGGATT[C3 spacer]-3′ (Underlined letters indicate the 5′-tagging portion of PTO)

The reaction was conducted in the final volume of 20 μl containing 100pg of genomic DNA of NG, 100 pg of genomic DNA of SA, 10 pmole of eachdownstream primer (SEQ ID NOs: 10 and 13), 10 pmole of each upstreamprimer (SEQ ID NOs: 2 and 14), 5 pmole of each PTO (SEQ ID NOs: 7 and15), 2 pmole of each CTO (SEQ ID NOs: 4 and 16), and 10 μl of 2× MasterMix containing 2-5 mM MgCl₂, 200 μM of dNTPs and 1.6 units of H-Taq DNApolymerase (Solgent, Korea); the tube containing the reaction mixturewas placed in the real-time thermocycler (CFX96, Bio-Rad); the reactionmixture was denatured for 15 min at 95□ and subjected to 60 cycles of 30sec at 95□, 60 sec at 60□, 30 sec at 72□. After the reaction, meltingcurve was obtained by cooling the reaction mixture to 35° C., holding atfor 35° C. for 30 sec, and heating slowly at 35° C. to 90° C. Thefluorescence was measured continuously during the temperature rise tomonitor dissociation of double-stranded DNAs. Melting peak was derivedfrom the melting curve data.

As shown in FIG. 20, multiple target signals (NG's Tm: 75.5° C. and SA'sTm: 63.5° C.) were detected in the presence of the templates. No signalwas detected in the absence of the templates.

These results indicate that PTOCE assay comprising melting analysisallows us to detect multiple target nucleic acids by using the same typeof a reporter molecule (e.g. FAM) in the condition that the extendedduplexes corresponding to the target nucleic acids have different T_(m)values.

Example 5 Evaluation of PTOCE Assay Comprising Melting Analysis onMicroarray

We further examined PTOCE assay comprising melting analysis onmicroarray. PTO cleavage was conducted in a separate vessel and analiquot of the resultant was taken into a microarray where CTO wasimmobilized. After the extension reaction, the presence of the extendedduplex was detected by melting analysis.

Taq DNA polymerase having 5′ nuclease activity was used for theextension of upstream primer, the cleavage of PTO and the extension ofPTO fragment. The extended duplex formed during the assay was designedto have a single label. The single label in the extended duplex wasprovided by PTO labeled with Quasar570 as a fluorescent reportermolecule at its 5′-end. PTO and CTO are blocked with a carbon spacer attheir 3′-ends. The CTO has poly(T)₅ as a linker arm and was immobilizedon the surface of a glass slide by using an amino group (AminnoC7) atits 5′-end. A marker probe having a fluorescent reporter molecule(Quasar570) at its 5′-end was immobilized on the surface of the glassslide by using an amino group at its 3′-end.

The sequences of synthetic template, upstream primer, PTO, CTO andmarker used in this Example are:

NG-T (SEQ ID NO: 1) 5′-AAATATGCGAAACACGCCAATGAGGGGCATGATGCTTTCTTTTTGTTCTTGCTCGGCAGAGCGAGTGATACCGATCCATTGAAAAA-3′ NG-R (SEQ ID NO: 2)5′-CAATGGATCGGTATCACTCGC-3′ NG-PTO-5 (SEQ ID NO: 17)5′-[Quasar570]ACGACGGCTTGGCTTTACTGCCCCTCATTGGCG TGTTTCG[C3 spacer]-3′NG-CTO-S1 (SEQ ID NO: 18)5′-[AminoC7]TTTTTCCTCCTCCTCCTCCTCCTCCTCCAGTAAAGCCAAGCCGTCGT[C3 Spacer]-3′ Marker (SEQ ID NO: 19)5′-[Quasar570]ATATATATAT[AminoC7]-3′(Underlined letters indicate the 5′-tagging portion of PTO)

NSB9 NHS slides (NSBPOSTECH, Korea) were used for fabrication of the CTOand marker (SEQ ID NOs: 18 and 19). The CTO and marker dissolved in NSBspotting buffer at the final concentration of 10 μM were printed on theNSB9 NHS slides with PersonalArrayer™ 16 Microarray Spotter (CapitalBio,China). The CTO and marker were spotted side by side in a 2×1 format(duplicate spots), and the resulting microarray was incubated in achamber maintained at ˜85% humidity for overnight. The slides were thenwashed in a buffer solution containing 2×SSPE (0.3 M sodium chloride,0.02 M sodium hydrogen phosphate and 2.0 mM EDTA), pH 7.4 and 7.0 mM SDSat 37° C. for 30 min to remove the non-specifically bound CTO and markerand rinsed with distilled water. Then, the DNA-functionalized slideswere dried using a slide centrifuge and stored in dark at 4° C. untiluse.

The cleavage reaction was conducted in the final volume of 50 μlcontaining 2 pmole of synthetic template (SEQ ID NO: 1) for NG gene, 10pmole of upstream primer (SEQ ID NO: 2), 1 pmole of PTO (SEQ ID NO: 17),and 25 μl of 2× Master Mix containing 2.5 mM MgCl₂, 200 μM of dNTPs, and4 units of H-Taq DNA polymerase (Solgent, Korea); the tube containingthe reaction mixture was placed in the real-time thermocycler (CFX96,Bio-Rad); the reaction mixture was denatured for 15 min at 95° C. andsubjected to 30 cycles of 30 sec at 95° C., 60 sec at 63° C.

The 30 μl of the resulting mixture was applied to a chamber assembled onthe surface of NSB glass slide on which the CTO (SEQ ID NO: 18) wascross-linked. The slide was placed on in situ block in a thermocycler(GenePro B4I, China). Six same slides were prepared for meltinganalysis. The extension reaction was allowed for 20 min at 55° C. Then,the resulting slides were incubated for 1 min at room temperature.Finally each slide was washed in distilled water for 1 min at 44° C.,52° C., 60□, 68□, 76□ or 84□. The image acquisition was carried out bythe use of Confocal Laser Scanner, Axon GenePix4100A (Molecular Device,US) with scanning at 5 μm pixel resolution. The fluorescence intensitywas analyzed by the use of quantitative microarray analysis software,GenePix pro6.0 software (Molecular Device, US). The fluorescenceintensity was expressed as spot-medians after local backgroundsubtractions. Each spot was duplicated for the test of reproducibility.The fluorescence intensity indicates the average value of the duplicatedspots.

As shown in FIGS. 21A and 21B, melting curve was obtained by measuringthe fluorescent intensity from the spots prepared by different washingtemperatures. The presence of the extended duplex was determined fromthe melting curve data.

Example 6 Evaluation of PTOCE Assay Comprising Real-Time Detection onMicroarray

We further examined PTOCE assay comprising real-time detection at apre-determined temperature on microarray.

Cleavage of PTO and extension of PTO fragment were repeated on amicroarray where CTO was immobilized. The presence of the extendedduplex was detected at a predetermined temperature in several determinedcycles.

Taq DNA polymerase having 5′ nuclease activity was used for theextension of upstream primer, the cleavage of PTO and the extension ofPTO fragment.

The extended duplex formed during the assay was designed to have asingle label or an interactive dual label. The single label in theextended duplex was provided by PTO labeled with a reporter molecule(reporter-labeled PTO). The interactive dual label in the extendedduplex was provided by CTO labeled with a reporter molecule and aquencher molecule (dual-labeled CTO). PTO and CTO are blocked with acarbon spacer at their 3′-ends.

The CTO has poly(T) as a linker arm. The CTO was immobilized on a glassslide by using an amino group (AminnoC7) at its 5′-end or its 3′-end. Amarker probe having a fluorescent reporter molecule (Quasar570) at its5′-end was immobilized on the glass slide by using an amino group at its3′-end. A fluorescent intensity on the glass slide was measured at apre-determined temperature. The detection temperature was determined tothe extent that the extended duplex maintains a double-stranded form.Synthetic oligonucleotide for Neisseria gonorrhoeae (NG) was used astemplates.

6-1. PTOCE Assay Using a Reporter-Labeled PTO

PTO has Quasar570 as a fluorescent reporter molecule at its 5′-end. TheCTO was immobilized through its 5′-end. In this labeling method, thedetection temperature was determined to the extent that the extendedduplex maintains a double-stranded form and the temperature is higherthan the T_(m) value of a hybrid between uncleaved PTO and CTO.

The sequences of synthetic template, upstream primer, PTO, CTO andmarker used in this Example are:

NG-T (SEQ ID NO: 1) 5′-AAATATGCGAAACACGCCAATGAGGGGCATGATGCTTTCTTTTTGTTCTTGCTCGGCAGAGCGAGTGATACCGATCCATTGAAAAAA-3′ NG-R (SEQ ID NO: 2)5′-CAATGGATCGGTATCACTCGC-3′ NG-PTO-5 (SEQ ID NO: 17)5′-[Quasar570]ACGACGGCTTGGCTTTACTGCCCCTCATTGGCG TGTTTCG[C3 spacer]-3′NG-CTO-S1 (SEQ ID NO: 18)5′-[AminoC7]TTTTTCCTCCTCCTCCTCCTCCTCCTCCAGTAAAGCCAAGCCGTCGT[C3 Spacer]-3′ Marker (SEQ ID NO: 19)5′-[Quasar570]ATATATATAT[AminoC7]-3′(Underlined letters indicate the 5′-tagging portion of PTO)

Slide preparation was conducted as the same protocol used in Example 5.

The PTOCE reaction was conducted in the final volume of 30 μl containing2 pmole of synthetic template (SEQ ID NO: 1) for NG gene, 10 pmole ofupstream primer (SEQ ID NO: 2), 1 pmole of PTO (SEQ ID NO: 17), and 15μl of 2× Master Mix containing 2.5 mM MgCl₂, 200 μM of dNTPs, and 2.4units of H-Taq DNA polymerase (Solgent, Korea); the whole mixture wasapplied to a chamber assembled on the surface of NSB glass slide onwhich the CTO (SEQ ID NO: 18) was cross-linked. The slide was placed onin situ block in a thermocycler (GenePro B4I, China). Five same slideswere prepared for cycling analysis. The PTOCE reaction was carried outas follows: 15 min denaturation at 95□ and 0, 5, 10, 20 or 30 cycles of30 sec at 95□, 60 sec at 60□, 60 sec at 55□. After the reaction of thecorresponding cycle number, the slides were washed in distilled water at64□ for 1 min. The image acquisition was carried out after each washingby the use of Confocal Laser Scanner, Axon GenePix4100A (MolecularDevice, US) with scanning at 5-μm pixel resolution. The fluorescenceintensity was analyzed by the use of quantitative microarray analysissoftware, GenePix pro6.0 software (Molecular Device, US). Thefluorescence intensity was expressed as spot-medians after localbackground subtractions. Each spot was duplicated for the test ofreproducibility. The fluorescence intensity indicates the average valueof the duplicated spots.

As shown in FIGS. 22A and 22B, the fluorescent intensity for the targetnucleic acid sequence was increased depending on cycle numbers (0cycle_RFU: 1,304±0.7; 5 cycles_RFU: 18,939±1,342.1; 10 cycles_RFU:30,619±285.0; 20 cycles_RFU: 56,248±2,208.3; and 30 cycles_RFU:64,645±1,110.2) in the presence of the template. There was no change ofthe fluorescent intensity depending on cycle numbers in the absence ofthe template.

6.2. PTOCE Assay Using a Dual-Labeled CTO

The CTO was immobilized through its 3′-end and has a quencher molecule(BHQ-2) and a fluorescent reporter molecule (Quasar570) in itstemplating portion.

The sequences of synthetic template, upstream primer, PTO, CTO andmarker used in this Example are:

NG-T (SEQ ID NO: 1) 5′-AAATATGCGAAACACGCCAATGAGGGGCATGATGCTTTCTTTTTGTTCTTGCTCGGCAGAGCGAGTGATACCGATCCATTGAAAAA-3′ NG-R (SEQ ID NO: 2)5′-CAATGGATCGGTATCACTCGC-3′ NG-PTO-6 (SEQ ID NO: 20)5′-ACGACGGCTTGGCTTTACTGCCCCTCATTGGCGTGTTTCG[C3 spacer]-3′ NG-CTO-S2(SEQ ID NO: 21) 5′-[BHQ-2]CCTCCTCCTCCTCCTCCTCC[T(Quasar570)]CCAGTAAAGCCAAGCCGTCGTTTTTTTTTTT[AminoC7]-3′ Marker (SEQ ID NO: 19)5′-[Quasar570]ATATATATAT[AminoC7]-3′(Underlined letters indicate the 5′tagging portion of PTO)

Slide preparation was conducted as the same protocol used in Example 5.

The PTOCE reaction was conducted in the final volume of 30 μl containing2 pmole of synthetic template (SEQ ID NO: 1) for NG gene, 10 pmole ofupstream primer (SEQ ID NO: 2), 1 pmole of PTO (SEQ ID NO: 20), and 15μl of 2× Master Mix containing 2.5 mM MgCl₂, 200 μM of dNTPs, and 2.4units of H-Taq DNA polymerase (Solgent, Korea); the whole mixture wasapplied to a chamber assembled on the surface of NSB glass slide onwhich the CTO was cross-linked (SEQ ID NO: 21). The slide was placed onin situ block in a thermocycler (GenePro B4I, China). Five same slideswere prepared for cycling analysis. The PTOCE reaction was carried outas follows: 15 min denaturation at 95□ and 0, 5, 10, 20 or 30 cycles of30 sec at 95□, 60 sec at 60□, 60 sec at 50□. After the reaction of thecorresponding cycle number, the image acquisition was carried out by theuse of Confocal Laser Scanner, Axon GenePix4100A (Molecular Device, US)with scanning at 5 μm pixel resolution. The fluorescence intensity wasanalyzed by the use of quantitative microarray analysis software,GenePix pro6.0 software (Molecular Device, US). The fluorescenceintensity was expressed as spot-medians after local backgroundsubtractions. Each spot was duplicated for the test of reproducibility.The fluorescence intensity indicates the average value of the duplicatedspots.

As shown in FIGS. 23A and 23B, the fluorescent intensity for the targetnucleic acid sequence was increased depending on cycle numbers (0cycle_RFU: 28,078±460.3; 5 cycles_RFU: 35,967±555.1; 10 cycles_RFU:44,674±186.0; 20 cycles_RFU: 65,423±2.1; and 30 cycles_RFU: 65,426±2.8)in the presence of template. There was no change of the fluorescentintensity depending on cycle numbers in the absence of the template.

Example 7 Detection of Multiple Target Nucleic Acid Sequences by PTOCEAssay Comprising End-Point Detection at a Pre-Determined Temperature onMicroarray

We further examined multiple target detection by PTOCE assay comprisingend-point detection at a pre-determined temperature on microarray.

PTO cleavage was conducted in a separate vessel with PCR process and analiquot of the resultant was taken into a microarray where CTO wasimmobilized. After extension reaction, the presence of the extendedduplex was detected by end-point detection at a pre-determinedtemperature.

Taq DNA polymerase having 5′ nuclease activity was used for theextension of upstream primer and downstream primer, the cleavage of PTOand the extension of PTO fragment.

The extended duplex formed during the assay was designed to have asingle label. The single label in the extended duplex was provided byPTO labeled with Quasar570 as a fluorescent reporter molecule at the5′-end of the PTO. PTO and CTO are blocked with a carbon spacer at their3′-ends.

The CTO has poly(T)₅ as a linker arm and was immobilized on a glassslide by using an amino group (AminnoC7) at its 5′-end. A marker probehaving a fluorescent reporter molecule (Quasar570) at its 5′-end wasimmobilized on the glass slide by using an amino group at its 3′-end.

A fluorescent intensity on the glass slide was measured at apre-determined temperature. The detection temperature was determined tothe extent that the extended duplex maintains a double-stranded form andthe temperature is higher than the T_(m) value of a hybrid betweenuncleaved PTO and CTO. Genomic DNAs of Staphylococcus aureus (SA) andNeisseria gonorrhoeae (NG) were used.

The sequences of upstream primer, downstream primer, PTO, CTO and markerused in this Example are:

NG-F (SEQ ID NO: 10) 5′-TACGCCTGCTACTTTCACGCT-3′ NG-R (SEQ ID NO: 2)5′-CAATGGATCGGTATCACTCGC-3′ NG-PTO-5 (SEQ ID NO: 17)5′-[Quasar570]ACGACGGCTTGGCTTTACTGCCCCTCATTGGCG TGTTTCG[C3 spacer]-3′NG-CTO-S1 (SEQ ID NO: 18)5′-[AminoC7]TTTTTCCTCCTCCTCCTCCTCCTCCTCCAGTAAAGCCAAGCCGTCGT[C3 Spacer]-3′ SA-F (SEQ ID NO: 13)5′-TGTTAGAATTTGAACAAGGATTTAATC-3′ SA-R2 (SEQ ID NO: 22)5′-TTAGCTCCTGCTCCTAAACCA-3′ SA-PTO-2 (SEQ ID NO: 23)5′-[Quasar570]AATCCGACCACGCTATGCTCATTCCGTGGTCAATCATTCGGTTTACG[C3 spacer]-3′ SA_CTO-S1 (SEQ ID NO: 24)5′-[AminoC7]TTTTTCTTCTTCTTCTTCTTCTTCTTCTTCCCCCAGCATAGCGTGGTCGGATT[C3 Spacer]-3′ Marker (SEQ ID NO: 19)5′-[Quasar570]ATATATATAT[AminoC7]-3′(Underlined letters indicated the 5′-tagging portion of PTO)

Slide preparation was conducted as the same protocol used in Example 5.

The cleavage reaction was conducted in the final volume of 50 μlcontaining each 100 pg genomic DNA of SA and/or NG, each 10 pmole ofdownstream primer (SEQ ID NOs: 10 and/or 13), each 10 pmole of upstreamprimer (SEQ ID NOs: 2 and/or 22), each pmole of PTO (SEQ ID NOs: 17and/or 23), and 25 μl of 2× Master Mix containing 2.5 mM MgCl₂, 200 μMof dNTPs, and 4 units of H-Taq DNA polymerase (Solgent, Korea); the tubecontaining the reaction mixture was placed in the real-time thermocycler(CFX96, Bio-Rad); the reaction mixture was denatured for 15 min at 95□and subjected to 60 cycles of 30 sec at 95□, 60 sec at 63□. The 30 μl ofthe resulting mixture was applied to a chamber assembled on the surfaceof NSB glass slide on which the CTOs (SEQ ID NOs: 18 and 24) werecross-linked. The slide was placed on in situ block in a thermocycler(GenePro B4I, China). The extension reaction was allowed for 20 min at55□. Then the slides were washed in distilled water at 64□ for 1 min.The image acquisition was carried out after each washing by the use ofConfocal Laser Scanner, Axon GenePix4100A (Molecular Device, US) withscanning at 10 μm pixel resolution. The fluorescence intensity wasanalyzed by the use of quantitative microarray analysis software,GenePix pro6.0 software (Molecular Device, US). The fluorescenceintensity was expressed as spot-medians after local backgroundsubtractions. Each spot was duplicated for the test of reproducibility.The fluorescence intensity indicates the average value of the duplicatedspots.

As shown in FIG. 24, the target signal for SA (RFU: 65,192±198.7) wasdetected in the presence of SA template. The target signal for NG (RFU:65,332±1.4) was detected in the presence of NG template. Both targetsignals for SA (RFU: 65,302±0.7) and NG (RFU 65,302±0.7) were detectedin the presence of both templates.

Having described a preferred embodiment of the present invention, it isto be understood that variants and modifications thereof falling withinthe spirit of the invention may become apparent to those skilled in thisart, and the scope of this invention is to be determined by appendedclaims and their equivalents.

What is claimed is:
 1. A method for detecting a target nucleic acidsequence from a DNA or a mixture of nucleic acids by a PTOCE (PTOCleavage and Extension) assay in a liquid phase, comprising: (a)hybridizing the target nucleic acid sequence with an upstreamoligonucleotide and a PTO (Probing and Tagging Oligonucleotide):wherein: the upstream oligonucleotide comprises a hybridizing nucleotidesequence complementary to the target nucleic acid sequence; the PTOcomprises (i) a 3′-targeting portion comprising a hybridizing nucleotidesequence complementary to the target nucleic acid sequence and (ii) a5′-tagging portion comprising a nucleotide sequence non-complementary tothe target nucleic acid sequence; the 3′-targeting portion is hybridizedwith the target nucleic acid sequence and the 5′-tagging portion is nothybridized with the target nucleic acid sequence; and the upstreamoligonucleotide is located upstream of the PTO; (b) contacting theresultant of the step (a) to an enzyme having a 5′ nuclease activityunder conditions for cleavage of the PTO; wherein the upstreamoligonucleotide or its extended strand induces cleavage of the PTO bythe enzyme having the 5′ nuclease activity such that the cleavagereleases a fragment comprising the 5′-tagging portion or a part of the5′-tagging portion of the PTO; (c) hybridizing the fragment releasedfrom the PTO with a CTO (Capturing and Templating Oligonucleotide);wherein the CTO comprises in a 3′ to 5′ direction (i) a capturingportion comprising a nucleotide sequence complementary to the 5′-taggingportion or a part of the 5′-tagging portion of the PTO and (ii) atemplating portion comprising a nucleotide sequence non-complementary tothe 5′-tagging portion and the 3′-targeting portion of the PTO; andwherein the fragment released from the PTO is hybridized with thecapturing portion of the CTO; (d) performing an extension reaction usingthe resultant of the step (c) and a template-dependent nucleic acidpolymerase; wherein the fragment hybridized with the capturing portionof the CTO is extended and an extended duplex is formed; wherein theextended duplex has a Tm value adjustable by (i) a sequence and/orlength of the fragment, (ii) a sequence and/or length of the CTO or(iii) the sequence and/or length of the fragment and the sequence and/orlength of the CTO; and wherein the extended duplex provides a targetsignal by (i) at least one label linked to the fragment and/or CTO, (ii)a label incorporated into the extended duplex during the extensionreaction, (iii) at least one label linked to the fragment and/or CTO anda label incorporated into the extended duplex during the extensionreaction or (iv) an intercalating label; and (e) detecting the extendedduplex by measuring the target signal in a liquid phase at apredetermined temperature that the extended duplex maintains itsdouble-stranded form, whereby the presence of the extended duplexindicates the presence of the target nucleic acid sequence; wherein theextended duplex is not immobilized on a solid substrate.
 2. The methodaccording to claim 1, wherein the target signal provided by the extendedduplex is given during the extension of the step (d); and wherein ahybrid between an uncleaved PTO and the CTO does not provides anon-target signal.
 3. The method according to claim 1: wherein thetarget signal provided by the extended duplex is given by thehybridization of the fragment and the CTO in the step (c) and theformation of the extended duplex maintains the target signal in the step(d); wherein a hybrid between an uncleaved PTO and the CTO does providea non-target signal; and wherein the predetermined temperature issufficient to dissociate the hybrid to remove the non-target signal. 4.The method according to claim 1, wherein the target signal is providedby at least one label linked to the fragment and/or the CTO.
 5. Themethod according to claim 4: wherein the fragment has an interactivedual label comprising a reporter molecule and a quencher molecule; andwherein the hybridization of the fragment and the CTO in the step (c)induces change of a signal from the interactive dual label to give thetarget signal and the extended duplex maintains the target signal. 6.The method according to claim 4: wherein the CTO has an interactive duallabel comprising a reporter molecule and a quencher molecule; andwherein the hybridization of the fragment and the CTO in the step (c)induces change of a signal from the interactive dual label to give thetarget signal and the extended duplex maintains the target signal. 7.The method according to claim 4: wherein the CTO has an interactive duallabel comprising a reporter molecule and a quencher molecule; andwherein the extension of the fragment in the step (d) induces change ofa signal from the interactive dual label to give the target signal. 8.The method according to claim 4: wherein the fragment has one of aninteractive dual label comprising a reporter molecule and a quenchermolecule and the CTO has the other of the interactive dual label; andwherein the hybridization of the fragment and the CTO in the step (c)induces change of a signal from the interactive dual label to give thetarget signal and the extended duplex maintains the target signal. 9.The method according to claim 4, wherein the fragment or the CTO has asingle label and the hybridization of the fragment and the CTO in thestep (c) induces change of a signal from the single label to give thetarget signal and the extended duplex maintains the target signal. 10.The method according to claim 4, wherein the CTO has a single label andthe extension of the fragment in the step (d) induces change of a signalfrom the single label to give the target signal.
 11. The methodaccording to claim 4, wherein the labels are positioned to the extentthat when a hybrid between an uncleaved PTO and the CTO is formed, thehybrid does not give a non-target signal in the step (d).
 12. The methodaccording to claim 4: wherein the labels are positioned to the extentthat when a hybrid between an uncleaved PTO and the CTO is formed, thehybrid gives a non-target signal in the step (d); and wherein the Tmvalue of the extended duplex is higher than that of the hybrid betweenthe uncleaved PTO and the CTO.
 13. The method according to claim 1:wherein the target signal is provided by a single label incorporatedinto the extended duplex during the extension reaction; wherein theincorporated single label is linked to a nucleotide incorporated duringthe extension reaction; and wherein the extension of the fragment in thestep (d) induces change of a signal from the single label to give thetarget signal in the step (d).
 14. The method according to claim 13,wherein the nucleotide incorporated during the extension reaction has afirst non-natural base and the CTO has a nucleotide having a secondnon-natural base with a specific binding affinity to the firstnon-natural base.
 15. The method according to claim 1: wherein thetarget signal is provided by a label incorporated into the extendedduplex during the extension reaction and a label linked to the fragmentand/or the CTO; wherein the label incorporated is linked to a nucleotideincorporated during the extension reaction; wherein the two labels arean interactive dual label of a reporter molecule and a quenchermolecule; and wherein the extension of the fragment in the step (d)induces change of a signal from the interactive dual label to give thetarget signal.
 16. The method according to claim 15, wherein thenucleotide incorporated during the extension reaction has a firstnon-natural base and the CTO has a nucleotide having a secondnon-natural base with a specific binding affinity to the firstnon-natural base.
 17. The method according to claim 1, wherein theupstream oligonucleotide is a primer.
 18. The method according to claim1, wherein the method further comprises repeating the steps (a)-(b),(a)-(d) or (a)-(e) with denaturation between repeating cycles.
 19. Themethod according to claim 1, wherein the steps (a)-(e) are performed inone or more reaction vessels.
 20. The method according to claim 1:wherein the method is performed to detect at least two types of targetnucleic acid sequences; and wherein the upstream oligonucleotidecomprises at least two types of oligonucleotides, the PTO comprises atleast two types of the PTOs, and the CTO comprises at least one type ofthe CTO.
 21. The method according to claim 1, wherein the method isperformed in the presence of a downstream primer.
 22. The methodaccording to claim 1, wherein the upstream oligonucleotide is a probe.